U.S. patent application number 13/090246 was filed with the patent office on 2012-02-09 for monomethylvaline compounds capable of conjugation to ligands.
This patent application is currently assigned to SEATTLE GENETICS, INC.. Invention is credited to SVETLANA O. DORONINA, ALLEN J. EBENS, TONI BETH KLINE, PAUL POLAKIS, PETER D. SENTER, MARK X. SLIWKOWSKI, SUSAN D. SPENCER, BRIAN E. TOKI.
Application Number | 20120034246 13/090246 |
Document ID | / |
Family ID | 34916511 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120034246 |
Kind Code |
A1 |
DORONINA; SVETLANA O. ; et
al. |
February 9, 2012 |
MONOMETHYLVALINE COMPOUNDS CAPABLE OF CONJUGATION TO LIGANDS
Abstract
Auristatin peptides, including MeVal-Val-Dil-Dap-Norephedrine
(MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), were prepared and attached
to Ligands through various linkers, including
maleimidocaproyl-val-cit-PAB. The resulting ligand drug conjugates
were active in vitro and in vivo.
Inventors: |
DORONINA; SVETLANA O.;
(SNOHOMISH, WA) ; SENTER; PETER D.; (SEATTLE,
WA) ; TOKI; BRIAN E.; (SHORELINE, WA) ; EBENS;
ALLEN J.; (SAN CARLOS, CA) ; KLINE; TONI BETH;
(SEATTLE, WA) ; POLAKIS; PAUL; (BURLINGAME,
CA) ; SLIWKOWSKI; MARK X.; (SAN CARLOS, CA) ;
SPENCER; SUSAN D.; (TIBURON, CA) |
Assignee: |
SEATTLE GENETICS, INC.
BOTHELL
WA
|
Family ID: |
34916511 |
Appl. No.: |
13/090246 |
Filed: |
April 19, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11833959 |
Aug 3, 2007 |
7964566 |
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13090246 |
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10983340 |
Nov 5, 2004 |
7498298 |
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11833959 |
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60622455 |
Oct 27, 2004 |
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60598899 |
Aug 4, 2004 |
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60557116 |
Mar 26, 2004 |
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60518534 |
Nov 6, 2003 |
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Current U.S.
Class: |
424/179.1 ;
424/178.1; 530/391.7; 530/391.9 |
Current CPC
Class: |
A61P 31/12 20180101;
Y02A 50/30 20180101; A61P 35/00 20180101; A61K 38/00 20130101; A61P
37/02 20180101; A61P 37/06 20180101; A61K 47/6811 20170801; A61K
47/6855 20170801; A61P 35/02 20180101; A61P 31/00 20180101; A61P
43/00 20180101; Y10T 428/13 20150115; A61K 47/6849 20170801; A61K
47/6851 20170801; C07K 2317/24 20130101; A61K 47/6889 20170801;
C07K 7/02 20130101; C07K 16/32 20130101; A61P 37/00 20180101; A61K
47/6803 20170801; A61P 31/04 20180101; A61K 2039/505 20130101 |
Class at
Publication: |
424/179.1 ;
530/391.7; 530/391.9; 424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C07K 16/30 20060101
C07K016/30 |
Claims
1. An antibody-drug conjugate compound comprising an antibody
covalently attached to one or more drug moieties, the compound
having Formula Ic: Ab A.sub.a-W.sub.w-Y.sub.y-D).sub.p Ic or a
pharmaceutically acceptable salt or solvate thereof, wherein: Ab is
an antibody which binds to the polypeptide of SEQ ID NO:5, A is a
Stretcher unit, a is 0 or 1, each W is independently an Amino Acid
unit, w is an integer ranging from 0 to 12, Y is a Spacer unit, and
y is 0, 1 or 2, p ranges from 1 to 20, and D is a drug moiety of
Formula D.sub.E: ##STR00087## wherein the wavy line of D.sub.E
indicates the covalent attachment site to A, W, or Y, and
independently at each location: R.sup.2 is selected from H and
C.sub.1-C.sub.8 alkyl; R.sup.3 is selected from H, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8
alkyl-aryl, C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is selected from H,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle, aryl,
C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
carbocycle), C.sub.3-C.sub.8 heterocycle and C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.5 is selected from H and
methyl; or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5
and 6; R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8
is independently selected from H, OH, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8 alkyl); R.sup.9
is selected from H and C.sub.1-C.sub.8 alkyl; and R.sup.18 is
selected from --C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
carbocycle).
2. The antibody-drug conjugate compound of claim 1, wherein the
antibody is attached to the drug moiety through a cysteine residue
of the antibody.
3. The antibody-drug conjugate compound of claim 1, wherein p is 1
to 4.
4. The antibody-drug conjugate compound of claim 1, wherein p is 2
to 8.
5. The antibody-drug conjugate compound of claim 1, wherein p is 2
to 5.
6. The antibody-drug conjugate compound of claim 1, having the
formula: ##STR00088##
7. The antibody-drug conjugate compound of claim 6 having the
formula: ##STR00089##
8. The antibody-drug conjugate compound of claim 1, having the
formula: ##STR00090##
9. The antibody-drug conjugate compound of claim 8 having the
formula: ##STR00091##
10. The antibody-drug conjugate compound of claim 9 having the
formula: ##STR00092##
11. The antibody-drug conjugate compound of claim 1, wherein w is
an integer ranging from 2 to 12.
12. The antibody-drug conjugate compound of claim 11, wherein w is
2.
13. The antibody-drug conjugate compound of claim 12 wherein
W.sub.w is -valine-citrulline-.
14. The antibody-drug conjugate compound of claim 1 wherein A is
maleimidocaproyl and a is 1.
15. The antibody-drug conjugate compound of claim 1 wherein Y is
p-aminobenzyloxycarbonyl (PAB) and y is 1.
16. The antibody-drug conjugate compound of claim 1, wherein D has
the formula: ##STR00093## and the wavy line indicates the point of
attachment to Y, W, A or Ab.
17. The antibody-drug conjugate compound of claim 1, wherein the
antibody binds to a portion of the polypeptide of SEQ ID NO:5 that
is expressed on the cell surface.
18. The antibody-drug conjugate compound of claim 17, wherein the
antibody is a humanized antibody.
19. The antibody-drug conjugate compound of claim 1, wherein the
antibody is a monoclonal antibody.
20. The antibody-drug conjugate compound of claim 1, wherein the
antibody is a chimeric antibody.
21. The antibody-drug conjugate compound of claim 1, wherein the
antibody is a humanized antibody.
22. The antibody-drug conjugate compound of claim 1, wherein the
antibody is a Fab fragment.
23. The antibody-drug conjugate compound of claim 1, having the
formula: ##STR00094## wherein Val is valine, and Cit is
citrulline.
24. The antibody-drug conjugate compound of claim 23, wherein the
antibody binds to a portion of the polypeptide of SEQ ID NO:5 that
is expressed on the cell surface.
25. The antibody-drug conjugate compound of claim 24, wherein the
antibody is a humanized antibody.
26. The antibody-drug conjugate compound of claim 24, wherein p is
1 to 4.
27. A pharmaceutical composition comprising an effective amount of
the antibody-drug conjugate compound of claim 1, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable diluent, carrier or excipient.
28. The pharmaceutical composition of claim 27 further comprising a
therapeutically effective amount of a chemotherapeutic agent
selected from a tubulin-forming inhibitor, a topoisomerase
inhibitor, a DNA binder, and gemcitabine.
29. A pharmaceutical composition comprising an effective amount of
the antibody-drug conjugate compound of claim 24, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable diluent, carrier or excipient.
30. The pharmaceutical composition of claim 29 further comprising a
therapeutically effective amount of a chemotherapeutic agent
selected from a tubulin-forming inhibitor, a topoisomerase
inhibitor, a DNA binder, and gemcitabine.
31. An article of manufacture comprising: an antibody drug
conjugate compound of claim 1; a container; and a package insert or
label indicating that the compound can be used to treat cancer
characterized by the expression of SEQ ID NO:5 or a portion thereof
expressed on the cell surface.
32. An article of manufacture comprising: an antibody drug
conjugate compound of claim 24; a container; and a package insert
or label indicating that the compound can be used to treat cancer
characterized by the expression of SEQ ID NO:5 or a portion thereof
expressed on the cell surface.
Description
CONTINUITY
[0001] This application is a continuation of U.S. patent Ser. No.
11/833,959, filed Aug. 3, 2007, which is a divisional of U.S.
patent Ser. No. 10/983,340, filed Nov. 5, 2004, which claims the
benefit of U.S. Provisional Patent Application No. 60/622,455,
filed Oct. 27, 2004; U.S. Provisional Patent Application No.
60/598,899, filed Aug. 4, 2004; U.S. Provisional Patent Application
No. 60/557,116, filed Mar. 26, 2004; and 60/518,534, filed Nov. 6,
2003; the disclosures of which are incorporated by reference
herein.
[0002] Some of the subject matter in this application was developed
under a joint research agreement between Seattle Genetics, Inc. and
Genentech, Inc.
1. FIELD OF THE INVENTION
[0003] The present invention is directed to a Drug Compound and
more particularly to Drug-Linker-Ligand Conjugates, Drug-Linker
Compounds, and Drug-Ligand Conjugates, to compositions including
the same, and to methods for using the same to treat cancer, an
autoimmune disease or an infectious disease. The present invention
is also directed to antibody-drug conjugates, to compositions
including the same, and to methods for using the same to treat
cancer, an autoimmune disease or an infectious. disease. The
invention also relates to methods of using antibody-drug conjugate
compounds for in vitro, in situ, and in vivo diagnosis or treatment
of mammalian cells, or associated pathological conditions.
2. BACKGROUND OF THE INVENTION
[0004] Improving the delivery of drugs and other agents to target
cells, tissues and tumors to achieve maximal efficacy and minimal
toxicity has been the focus. of considerable research for many
years. Though many attempts have been made to develop effective
methods for importing biologically active molecules into cells,
both in vivo and in vitro, none has proved to be entirely
satisfactory. Optimizing the association of the drug with its
intracellular target, while minimizing intercellular redistribution
of the drug, e.g., to neighboring cells, is often difficult or
inefficient.
[0005] Most agents currently administered to a patient parenterally
are not targeted, resulting in systemic delivery of the agent to
cells and tissues of the body where it is unnecessary, and often
undesirable. This may result in adverse drug side effects, and
often limits the dose of a drug (e.g., chemotherapeutic
(anti-cancer), cytotoxic, enzyme inhibitor agents and antiviral or
antimicrobial drugs) that can be administered. By comparison,
although oral administration of drugs is considered to be a
convenient and economical mode of administration, it shares the
same concerns of non-specific toxicity to unaffected cells once the
drug has been absorbed into the systemic circulation. Further
complications involve problems with oral bioavailability and
residence of drug in the gut leading to additional exposure of gut
to the drug and hence risk of gut toxicities. Accordingly, a major
goal has been to develop methods for specifically targeting agents
to cells and tissues. The benefits of such treatment include
avoiding the general physiological effects of inappropriate
delivery of such agents to other cells and tissues, such as
uninfected cells. Intracellular targeting may be achieved by
methods, compounds and formulations which allow accumulation or
retention of biologically active agents, i.e. active metabolites,
inside cells.
[0006] Monoclonal antibody therapy has been established for the
targeted treatment of patients with cancer, immunological and
angiogenic disorders.
[0007] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg. Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278)
theoretically allows targeted delivery of the drug moiety to
tumors, and intracellular accumulation therein, while systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet
pp. (Mar. 15, 1986):603-05; Thorpe, 1985, "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., 1986, supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Kerr et al., 1997, Bioconjugate Chem.
8(6):781-784; Mandler et al. (2000) Jour. of the Nat. Cancer Inst.
92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem.
Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al.
(1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res.
53:3336-3342). The toxins may affect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition (Meyer, D. L. and Senter, P. D. "Recent
Advances in Antibody Drug Conjugates for Cancer Therapy" in Annual
Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237).
Some cytotoxic drugs tend to be inactive or less active when
conjugated to large antibodies or protein receptor ligands.
[0008] ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al.
(2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate
composed of a hu CD33 antibody linked to calicheamicin, was
approved in 2000 for the treatment of acute myeloid leukemia by
injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;
5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an
antibody drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an
antibody drug conjugate composed of the anti-prostate specific
membrane antigen (PSMA) monoclonal antibody linked to the
maytansinoid drug moiety, DM1, is under development for the
potential treatment of prostate tumors. The same maytansinoid drug
moiety, DM1, was linked through a non-disulfide linker, SMCC, to a
mouse murine monoclonal antibody, TA.1 (Chari et al. (1992) Cancer
Research 52:127-131). This conjugate was reported to be 200-fold
less potent than the corresponding disulfide linker conjugate. The
SMCC linker was considered therein to be "noncleavable."
[0009] Several short peptidic compounds have been isolated from the
marine mollusc Dolabella auricularia and found to have biological
activity (Pettit et al. (1993) Tetrahedron 49:9151; Nakamura et al.
(1995) Tetrahedron Letters 36:5059-5062; Sone et al. (1995) Jour.
Org. Chem. 60:4474). Analogs of these compounds have also been
prepared, and some were found to have biological activity (for a
review, see Pettit et al. (1998) Anti-Cancer Drug Design
13:243-277). For example, auristatin E (U.S. Pat. No. 5,635,483) is
a synthetic analogue of the marine natural product Dolastatin 10,
an agent that inhibits tubulin polymerization by binding to the
same domain on tubulin as the anticancer drug vincristine (G. R.
Pettit, (1997) Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin 10,
auristatin PE, and auristatin E are linear peptides having four
amino acids, three of which are unique to the dolastatin class of
compounds, and a C-terminal amide.
[0010] The auristatin peptides, auristain E (AE) and
monomethylauristatin (MMAE), synthetic analogs of dolastatin, were
conjugated to: (i) chimeric monoclonal antibodies cBR96 (specific
to Lewis Y on carcinomas); (ii) cAC10 which is specific to CD30 on
hematological malignancies (Klussman, et al. (2004), Bioconjugate
Chemistry 15(4):765-773; Doronina et al. (2003) Nature
Biotechnology 21(7):778-784; "Monomethylvaline Compounds Capable of
Conjugation to Ligands"; Francisco et al. (2003) Blood
102(4):1458-1465; U.S. Publication 2004/0018194; (iii) anti-CD20
antibodies such as RITUXAN.RTM. (WO 04/032828) for the treatment of
CD20-expressing cancers and immune disorders; (iv) anti-EphB2
antibodies 2119 and anti-IL-8 for treatment of colorectal cancer
(Mao, et al. (2004) Cancer Research 64(3):781-788); (v) E-selectin
antibody (Bhaskar et al. (2003) Cancer Res. 63:6387-6394); and (vi)
other anti-CD30 antibodies (WO 03/043583).
[0011] Auristatin E conjugated to monoclonal antibodies are
disclosed in Senter et al, Proceedings of the American Association
for Cancer Research, Volume 45, Abstract Number 623, presented Mar.
28, 2004.
[0012] Despite in vitro data for compounds of the dolastatin class
and its analogs, significant general toxicities at doses required
for achieving a therapeutic effect compromise their efficacy in
clinical studies. Accordingly, there is a clear need in the art for
dolastatin/auristatin derivatives having significantly lower
toxicity, yet useful therapeutic efficiency. These and other
limitations and problems of the past are addressed by the present
invention.
[0013] The ErbB family of receptor tyrosine kinases are important
mediators of cell growth, differentiation and survival. The
receptor family includes four distinct members including epidermal
growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of
anti-ErbB2 antibodies has been characterized using the human breast
tumor cell line SKBR3 (Hudziak et al., (1989) Mol. Cell. Biol.
9(3):1165-1172. Maximum inhibition was obtained with the antibody
called 4D5 which inhibited cellular proliferation by 56%. Other
antibodies in the panel reduced cellular proliferation to a lesser
extent in this assay. The antibody 4D5 was further found to
sensitize ErbB2-overexpressing breast tumor cell lines to the
cytotoxic effects of TNF-.alpha. (U.S. Pat. No. 5,677,171). The
anti-ErbB2 antibodies discussed in Hudziak et al. are further
characterized in Fendly et al. (1990) Cancer Research 50:1550-1558;
Kotts et al. (1990) In vitro 26(3):59A; Sarup et al. (1991) Growth
Regulation 1:72-82; Shepard et al. J. (1991) Clin. Immunol.
11(3):117-127; Kumar et al., (1991) Mol. Cell. Biol. 11(2):979-986;
Lewis et al. (1993) Cancer Immunol. Immunother. 37:255-263; Pietras
et al. (1994) Oncogene 9:1829-1838; Vitetta et al. (1994) Cancer
Research 54:5301-5309; Sliwkowski et al. (1994) J. Biol. Chem.
269(20):14661-14665; Scott et al. (1991) J. Biol. Chem.
266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994)
91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465; and
Schaefer et al. (1997) Oncogene 15:1385-1394.
[0014] Other anti-ErbB2 antibodies with various properties have
been described in Tagliabue et al. Int. J. Cancer 47:933-937
(1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al.
Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular
Carcinogenesis 3:350-362 (1990); Stancovski et al. Proc. Natl.
Acad. Sci. USA 88:8691-8695 (1991); Bacus et al. Cancer Research
52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993);
WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992);
Hancock et al. (1991) Cancer Res. 51:4575-4580; Shawver et al.
(1994) Cancer Res. 54:1367-1373; Arteaga et al. (1994) Cancer Res.
54:3758-3765; Harwerth et al. (1992) J. Biol. Chem.
267:15160-15167; U.S. Pat. No. 5,783,186; and Klapper et al. (1997)
Oncogene 14:2099-2109.
[0015] Homology screening has resulted in the identification of two
other ErbB receptor family members; ErbB3 (U.S. Pat. No. 5,183,884;
U.S. Pat. No. 5,480,968; Kra U. S. et al. (1989) Proc. Natl. Acad.
Sci. USA 86:9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993)
Proc. Natl. Acad. Sci. USA 90:1746-1750; and Plowman et al. (1993)
Nature 366:473-475). Both of these receptors display increased
expression on at least some breast cancer cell lines.
[0016] HERCEPTIN.RTM. (Trastuzumab) is a recombinant DNA-derived
humanized monoclonal antibody that selectively binds with high
affinity in a cell-based assay (Kd=5 nM) to the extracellular
domain of the human epidermal growth factor receptor2 protein, HER2
(ErbB2) (U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S.
Pat. No. 6,407,213; U.S. Pat. No. 6,639,055; Coussens L, et al.
(1985) Science 230:1132-9; Slamon D J, et al. (1989) Science
244:707-12). Trastuzumab is an IgG1 kappa antibody that contains
human framework regions with the complementarity-determining
regions of a murine antibody (4D5) that binds to HER2. Trastuzumab
binds to the HER2 antigen and thus. inhibits the growth of
cancerous cells. Because Trastuzumab is a humanized antibody, it
minimizes any HAMA response in patients. The humanized antibody
against HER2 is produced by a mammalian cell (Chinese Hamster
Ovary, CHO) suspension culture. The HER2 (or c-erbB2)
proto-oncogene encodes a transmembrane receptor protein of 185 kDa,
which is structurally related to the epidermal growth factor
receptor. HER2 protein overexpression is observed in 25%-30% of
primary breast cancers and can be determined using an
immunohistochemistry based assessment of fixed tumor blocks (Press
M F, et al. (1993) Cancer Res 53:4960-70. Trastuzumab has been
shown, in both in vitro assays and in animals, to inhibit the
proliferation of human tumor cells that overexpress HER2 (Hudziak R
M, et al. (1989) Mol Cell Biol 9:1165-72; Lewis G D, et al. (1993)
Cancer Immunol Immunother; 37:255-63; Baselga J, et al. (1998)
Cancer Res. 58:2825-2831). Trastuzumab is a mediator of
antibody-dependent cellular cytotoxicity, ADCC (Hotaling T E, et
al. (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res;
37:471; Pegram M D, et al. (1997) [abstract]. Proc Am Assoc Cancer
Res; 38:602). In vitro, Trastuzumab mediated ADCC has been shown to
be preferentially exerted on HER2 overexpressing cancer cells
compared with cancer cells that do not overexpress HER2.
HERCEPTIN.RTM. as a single agent is indicated for the treatment of
patients with metastatic breast cancer whose tumors overexpress the
HER2 protein and who have received one or more chemotherapy
regimens for their metastatic disease. HERCEPTIN.RTM. in
combination with paclitaxel is indicated for treatment of patients
with metastatic breast cancer whose tumors overexpress the HER2
protein and who have not received chemotherapy for their metastatic
disease. HERCEPTIN.RTM. is clinically active in patients with
ErbB2-overexpressing metastatic breast cancers that have received
extensive prior anti-cancer therapy (Baselga et al, (1996) J. Clin.
Oncol. 14:737-744).
[0017] The murine monoclonal anti-HER2 antibody inhibits the growth
of breast cancer cell lines that overexpress HER2 at the 2+ and 3+
(1-2.times.10.sup.6 HER2 receptors per cell) level, but has no
activity on cells that express lower levels of HER2 (Lewis et al.,
(1993) Cancer Immunol. Immunother. 37:255-263). Based on this
observation, antibody 4D5 was humanized (huMAb4D5-8, rhuMAb HER2,
U.S. Pat. No. 5,821,337; Carter et al., (1992) Proc. Natl. Acad.
Sci. USA 89: 4285-4289) and tested in breast cancer patients whose
tumors overexpress HER2 but who had progressed after conventional
chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17:
2639-2648).
[0018] Although HERCEPTIN is a breakthrough in treating patients
with ErbB2-overexpressing breast cancers that have received
extensive prior anti-cancer therapy, some patients in this
population fail to respond or respond only poorly to HERCEPTIN
treatment.
[0019] Therefore, there is a significant clinical need for
developing further HER2-directed cancer therapies for those
patients with HER2-overexpressing tumors or other diseases
associated with HER2 expression that do not respond, or respond
poorly, to HERCEPTIN treatment.
[0020] The recitation of any reference in this application is not
an admission that the reference is prior art to this
application.
3. SUMMARY OF THE INVENTION
[0021] In one aspect, the present invention provides
Drug-Linker-Ligand compounds having the Formula Ia:
L A.sub.a-W.sub.w-Y.sub.y-D).sub.p Ia
[0022] or a pharmaceutically acceptable salt or solvate thereof
[0023] wherein,
[0024] L- is a Ligand unit;
[0025] -A.sub.a-W.sub.w-Y.sub.y- is a Linker unit (LU), wherein the
Linker unit includes:
[0026] -A- is a Stretcher unit,
[0027] a is 0 or 1,
[0028] each -W- is independently an Amino Acid unit,
[0029] w is an integer ranging from 0 to 12,
[0030] -Y- is a Spacer unit, and
[0031] y is 0, 1 or 2;
[0032] p ranges from 1 to about 20; and
[0033] -D is a Drug unit having the Formulas D.sub.E and
D.sub.F:
##STR00001##
[0034] wherein, independently at each location:
[0035] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0036] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0037] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0038] R.sup.5 is selected from H and methyl;
[0039] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and
[0040] n is selected from 2, 3, 4, 5 and 6;
[0041] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0042] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0043] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0044] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0045] R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0046] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0047] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0048] m is an integer ranging from 1-1000;
[0049] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0050] R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
[0051] each occurrence of R.sup.15 is independently H, COON,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0052] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH; where; n is an
integer ranging from 0 to 6; and
[0053] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
carbocycle).
[0054] In another aspect, Drug Compounds having the Formula Ib are
provided:
##STR00002##
or pharmaceutically acceptable salts or solvates thereof,
[0055] wherein:
[0056] R.sup.2 is selected from hydrogen and --C.sub.1-C.sub.8
alkyl;
[0057] R.sup.3 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
[0058] R.sup.4 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 jointly, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are
attached;
[0059] R.sup.6 is selected from H and --C.sub.1-C.sub.8 alkyl;
[0060] R.sup.7 is selected from H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
[0061] each R.sup.8 is independently selected from H, --OH,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl);
[0062] R.sup.9 is selected from H and --C.sub.1-C.sub.8 alkyl;
[0063] R.sup.10 is selected from aryl group or --C.sub.3-C.sub.8
heterocycle;
[0064] Z is --O--, --S--, --NH--, or --NR.sup.12--, wherein
R.sup.12 is C.sub.1-C.sub.8 alkyl;
[0065] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
--C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0066] m is an integer ranging from 1-1000;
[0067] R.sup.13 is --C.sub.2-C.sub.8 alkyl;
[0068] R.sup.14 is H or --C.sub.1-C.sub.8 alkyl;
[0069] each occurrence of R.sup.15 is independently H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0070] each occurrence of R.sup.16 is independently H,
--C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH; and
[0071] n is an integer ranging from 0 to 6.
[0072] The compounds of Formula (Ib) are useful for treating
cancer, an autoimmune disease or an infectious disease in a patient
or useful as an intermediate for the synthesis of a Drug-Linker,
Drug-Linker-Ligand Conjugate, and Drug-Ligand Conjugate having a
cleavable Drug unit.
[0073] In another aspect, compositions are provided including an
effective amount of a Drug-Linker-Ligand Conjugate and a
pharmaceutically acceptable carrier or vehicle.
[0074] In still another aspect, the invention provides
pharmaceutical compositions comprising an effective amount of a
Drug-Linker Compound and a pharmaceutically acceptable carrier or
vehicle.
[0075] In still another aspect, the invention provides compositions
comprising an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate and a
pharmaceutically acceptable carrier or vehicle.
[0076] In yet another aspect, the invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell including administering to a patient in need thereof an
effective amount of a Drug-Linker Compound.
[0077] In another aspect, the invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell including administering to a patient in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
[0078] In another aspect, the invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell including administering to a patient in need thereof an
effective amount of a Drug-Ligand Conjugate having a cleavable Drug
unit from the Drug-Ligand Conjugate.
[0079] In still another aspect, the invention provides methods for
treating cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
[0080] In yet another aspect, the invention provides methods for
treating cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
[0081] In yet another aspect, the invention provides methods for
treating cancer including administering to a patient in need
thereof an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate.
[0082] In still another aspect, the invention provides methods for
killing or inhibiting the replication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
[0083] In another aspect, the invention provides methods for
killing or inhibiting the replication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
[0084] In another aspect, the invention provides methods for
killing or inhibiting the replication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate.
[0085] In yet another aspect, the invention provides methods for
treating an autoimmune disease including administering to a patient
in need thereof an effective amount of a Drug-Linker Compound.
[0086] In yet another aspect, the invention provides methods for
treating an autoimmune disease including administering to a patient
in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
[0087] In yet another aspect, the invention provides methods for
treating an autoimmune disease including administering to a patient
in need thereof an effective amount of a Drug-Ligand Conjugate
having a cleavable Drug unit from the Drug-Ligand Conjugate.
[0088] In still another aspect, the invention provides methods for
treating an infectious. disease including administering to a
patient in need thereof an effective amount of a Drug-Linker
Compound.
[0089] In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
[0090] In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Ligand Conjugate
having a cleavable Drug unit from the Drug-Ligand Conjugate.
[0091] In yet another aspect, the invention provides methods for
preventing the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker Compound.
[0092] In another aspect, the invention provides methods for
preventing the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker-Ligand Conjugate.
[0093] In another aspect, the invention provides methods for
preventing the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Ligand Conjugate having a cleavable Drug unit from
the Drug-Ligand Conjugate.
[0094] In still another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
[0095] In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
[0096] In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate.
[0097] In still another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
[0098] In another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
[0099] In another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate.
[0100] In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Linker
Compound.
[0101] In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
[0102] In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Ligand
Conjugate having a cleavable Drug unit from the Drug-Ligand
Conjugate.
[0103] In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Linker
Compound.
[0104] In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
[0105] In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Ligand
Conjugate having a cleavable Drug unit from the Drug-Ligand
Conjugate.
[0106] In another aspect, a Drug Compound is provided which can be
used as an intermediate for the synthesis of a Drug-Linker Compound
having a cleavable Drug unit from the Drug-Ligand Conjugate.
[0107] In another aspect, a Drug-Linker Compound is provided which
can be used as an intermediate for the synthesis of a
Drug-Linker-Ligand Conjugate.
[0108] In another aspect, compounds having Formula Ia' are
provided:
Ab A.sub.a-W.sub.w-Y.sub.y-D).sub.p Ia'
[0109] or a pharmaceutically acceptable salt or solvate thereof,
wherein: [0110] Ab includes an antibody including one which binds
to CD30, CD40, CD70, and Lewis Y antigen,
[0111] A is a Stretcher unit,
[0112] a is 0 or 1,
[0113] each W is independently an Amino Acid unit,
[0114] w is an integer ranging from 0 to 12,
[0115] Y is a Spacer unit, and
[0116] y is 0, 1 or 2,
[0117] p ranges from 1 to about 20, and
[0118] D is a Drug unit selected from Formulas D.sub.E and
D.sub.F:
##STR00003##
[0119] wherein, independently at each location:
[0120] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0121] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0122] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0123] R.sup.5 is selected from H and methyl;
[0124] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5
and 6;
[0125] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0126] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0127] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0128] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0129] R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0130] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0131] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0132] m is an integer ranging from 1-1000;
[0133] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0134] R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
[0135] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3-C.sub.1-C.sub.8 alkyl;
[0136] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH;
[0137] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
[0138] n is an integer ranging from 0 to 6.
[0139] In one embodiment, Ab is not an antibody which binds to an
ErbB receptor or which binds to one or more of receptors
(1)-(35):
[0140] (1) BMPR1B (bone morphogenetic protein receptor-type IB,
Genbank accession no. NM.sub.--001203);
[0141] (2) E16 (LAT1, SLC7A5, Genbank accession no.
NM.sub.--003486);
[0142] (3) STEAP1 (six transmembrane epithelial antigen of
prostate, Genbank accession no. NM.sub.--012449);
[0143] (4) 0772P (CA125, MUC16, Genbank accession no.
AF361486);
[0144] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
[0145] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family
34 (sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
[0146] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
[0147] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA
2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.
AY358628);
[0148] (9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
[0149] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
[0150] (11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1,
STEAP2, STMP, prostate cancer associated gene 1, prostate cancer
associated protein 1, six transmembrane epithelial antigen of
prostate 2, six transmembrane prostate protein, Genbank accession
no. AF455138);
[0151] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4, Genbank
accession no. NM.sub.--017636);
[0152] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor, Genbank accession no.
NP.sub.--003203 or NM.sub.--003212);
[0153] (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein
Barr virus receptor) or Hs.73792, Genbank accession no.
M26004);
[0154] (15) CD79b (IGb (immunoglobulin-associated beta), B29,
Genbank accession no. NM.sub.--000626);
[0155] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession
no. NM.sub.--030764);
[0156] (17) HER2 (Genbank accession no. M11730);
[0157] (18) NCA (Genbank accession no. M18728);
[0158] (19) MDP (Genbank accession no. BC017023);
[0159] (20) IL20R.alpha. (Genbank accession no. AF184971);
[0160] (21) Brevican (Genbank accession no. AF229053);
[0161] (22) Ephb2R (Genbank accession no. NM.sub.--004442);
[0162] (23) ASLG659 (Genbank accession no. AX092328);
[0163] (24) PSCA (Genbank accession no. AJ297436);
[0164] (25) GEDA (Genbank accession no. AY260763);
[0165] (26) BAFF-R (Genbank accession no. NP 443177.1);
[0166] (27) CD22 (Genbank accession no. NP-001762.1);
[0167] (28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation, Genbank
accession No. NP.sub.--001774.1);
[0168] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G
protein-coupled receptor that is activated by the CXCL13 chemokine,
functions in lymphocyte migration and humoral defense, plays a role
in HIV-2 infection and perhaps development of AIDS, lymphoma,
myeloma, and leukemia, Genbank accession No.
NP.sub.--001707.1);
[0169] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia
antigen) that binds peptides and presents them to CD4+ T
lymphocytes, Genbank accession No. NP.sub.--002111.1);
[0170] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel
5, an ion channel gated by extracellular ATP, may be involved in
synaptic transmission and neurogenesis, deficiency may contribute
to the pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
[0171] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2,
Genbank accession No. NP.sub.--001773.1);
[0172] (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane
protein of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
[0173] (34) FCRH1 (Fc receptor-like protein 1, a putative receptor
for the immunoglobulin Fc domain that contains C2 type Ig-like and
ITAM domains, may have a role in B-lymphocyte differentiation,
Genbank accession No. NP.sub.--443170.1); or
[0174] (35) IRTA2 (Immunoglobulin superfamily receptor
translocation associated 2, a putative immunoreceptor with possible
roles in B cell development and lymphomagenesis; deregulation of
the gene by translocation occurs in some B cell malignancies,
Genbank accession No. NP.sub.--112571.1).
[0175] In still another aspect, the invention provides
pharmaceutical compositions comprising an effective amount of a
Drug-Linker-Antibody Conjugate and a pharmaceutically acceptable
carrier or vehicle.
[0176] In still another aspect, the invention provides compositions
comprising an effective amount of a Drug-Antibody Conjugate having
a cleavable Drug unit (moiety) from the Drug-Antibody Conjugate and
a pharmaceutically acceptable carrier or vehicle.
[0177] In another aspect, the invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell including administering to a patient in need thereof an
effective amount of a Drug-Linker-Antibody Conjugate.
[0178] In another aspect, the invention provides methods for
killing or inhibiting the multiplication of a tumor cell or cancer
cell including administering to a patient in need thereof an
effective amount of a Drug-Antibody Conjugate having a cleavable
Drug unit from the Drug-Antibody Conjugate.
[0179] In yet another aspect, the invention provides methods for
treating cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
[0180] In yet another aspect, the invention provides methods for
treating cancer including administering to a patient in need
thereof an effective amount of a Drug-Antibody Conjugate having a
cleavable Drug unit from the Drug-Antibody Conjugate.
[0181] In another aspect, the invention provides methods for
killing or inhibiting the replication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
[0182] In another aspect, the invention provides methods for
killing or inhibiting the replication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Antibody Conjugate having a
cleavable Drug unit from the Drug-Antibody Conjugate.
[0183] In yet another aspect, the invention provides methods for
treating an autoimmune disease including administering to a patient
in need thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
[0184] In yet another aspect, the invention provides methods for
treating an autoimmune disease including administering to a patient
in need thereof an effective amount of a Drug-Antibody Conjugate
having a cleavable Drug unit from the Drug-Antibody Conjugate.
[0185] In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
[0186] In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Antibody Conjugate
having a cleavable Drug unit from the Drug-Antibody Conjugate.
[0187] In another aspect, the invention provides methods for
preventing the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker-Antibody Conjugate.
[0188] In another aspect, the invention provides methods for
preventing the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Antibody Conjugate having a cleavable Drug unit
from the Drug-Antibody Conjugate.
[0189] In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
[0190] In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Antibody Conjugate having a
cleavable Drug unit from the Drug-Antibody Conjugate.
[0191] In another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
[0192] In another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Antibody Conjugate having a
cleavable Drug unit from the Drug-Antibody Conjugate.
[0193] In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a
Drug-Linker-Antibody Conjugate.
[0194] In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Antibody
Conjugate having a cleavable Drug unit from the Drug-Antibody
Conjugate.
[0195] In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a
Drug-Linker-Antibody Conjugate.
[0196] In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Antibody
Conjugate having a cleavable Drug unit from the Drug-Antibody
Conjugate.
[0197] In another aspect, a Drug Compound is provided which can be
used as an intermediate for the synthesis of a Drug-Linker Compound
having a cleavable Drug unit from the Drug-Antibody Conjugate.
[0198] In another aspect, a Drug-Linker Compound is provided which
can be used as an intermediate for the synthesis of a
Drug-Linker-Antibody Conjugate.
[0199] In one aspect, the present invention provides
Drug-Linker-Antibody Conjugates (also referred to as antibody-drug
conjugates) having Formula Ic:
Ab A.sub.a-W.sub.w-Y.sub.y-D).sub.p Ic
[0200] or a pharmaceutically acceptable salt or solvate thereof,
wherein:
[0201] Ab is an antibody which binds to one or more of the antigens
(1)-(35):
[0202] (1) BMPR1B (bone morphogenetic protein receptor-type IB,
Genbank accession no. NM.sub.--001203);
[0203] (2) E16 (LAT1, SLC7A5, Genbank accession no.
NM.sub.--003486);
[0204] (3) STEAP1 (six transmembrane epithelial antigen of
prostate, Genbank accession no. NM.sub.--012449);
[0205] (4) 0772P (CA125, MUC16, Genbank accession no.
AF361486);
[0206] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
[0207] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family
34 (sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
[0208] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
[0209] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA
2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.
AY358628);
[0210] (9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
[0211] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
[0212] (11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1,
STEAP2, STMP, prostate cancer associated gene 1, prostate cancer
associated protein 1, six transmembrane epithelial antigen of
prostate 2, six transmembrane prostate protein, Genbank accession
no. AF455138);
[0213] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4, Genbank
accession no. NM.sub.--017636);
[0214] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor, Genbank accession no.
NP.sub.--003203 or NM.sub.--003212);
[0215] (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein
Barr virus receptor) or Hs.73792, Genbank accession no.
M26004);
[0216] (15) CD79b (1 Gb (immunoglobulin-associated beta), B29,
Genbank accession no. NM.sub.--000626);
[0217] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession
no. NM.sub.--030764);
[0218] (17) HER2 (Genbank accession no. M11730);
[0219] (18) NCA (Genbank accession no. M18728);
[0220] (19) MDP (Genbank accession no. BC017023);
[0221] (20) IL20R.alpha. (Genbank accession no. AF184971);
[0222] (21) Brevican (Genbank accession no. AF229053);
[0223] (22) Ephb2R (Genbank accession no. NM.sub.--004442);
[0224] (23) ASLG659 (Genbank accession no. AX092328);
[0225] (24) PSCA (Genbank accession no. AJ297436);
[0226] (25) GEDA (Genbank accession no. AY260763);
[0227] (26) BAFF-R (Genbank accession no. NP.sub.--443177.1);
[0228] (27) CD22 (Genbank accession no. NP-001762.1);
[0229] (28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated
alpha, a B cell-specific protein that covalently interacts with Ig
beta (CD79B) and forms a complex on the surface with Ig M
molecules, transduces a signal involved in B-cell differentiation,
Genbank accession No. NP.sub.--001774.1);
[0230] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G
protein-coupled receptor that is activated by the CXCL13 chemokine,
functions in lymphocyte migration and humoral defense, plays a role
in HIV-2 infection and perhaps development of AIDS, lymphoma,
myeloma, and leukemia, Genbank accession No.
NP.sub.--001707.1);
[0231] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia
antigen) that binds peptides and presents them to CD4+ T
lymphocytes, Genbank accession No. NP.sub.--002111.1);
[0232] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel
5, an ion channel gated by extracellular ATP, may be involved in
synaptic transmission and neurogenesis, deficiency may contribute
to the pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
[0233] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2,
Genbank accession No. NP.sub.--001773.1);
[0234] (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane
protein of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
[0235] (34) FCRH1 (Fc receptor-like protein 1, a putative receptor
for the immunoglobulin Fc domain that contains C2 type Ig-like and
ITAM domains, may have a role in B-lymphocyte differentiation,
Genbank accession No. NP 443170.1); or
[0236] (35) IRTA2 (Immunoglobulin superfamily receptor
translocation associated 2, a putative immunoreceptor with possible
roles in B cell development and lymphomagenesis; deregulation of
the gene by translocation occurs in some B cell malignancies,
Genbank accession No. NP.sub.--112571.1);
[0237] A is a Stretcher unit,
[0238] a is 0 or 1,
[0239] each W is independently an Amino Acid unit,
[0240] w is an integer ranging from 0 to 12,
[0241] Y is a Spacer unit, and
[0242] y is 0, 1 or 2,
[0243] p ranges from 1 to about 20, and
[0244] D is a Drug moiety selected from Formulas D.sub.E and
D.sub.F:
##STR00004##
[0245] wherein the wavy line of D.sub.E and D.sub.F indicates the
covalent attachment site to
[0246] A, W, or Y, and independently at each location:
[0247] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0248] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0249] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0250] R.sup.5 is selected from H and methyl;
[0251] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and
[0252] n is selected from 2, 3, 4, 5 and 6;
[0253] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0254] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0255] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0256] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0257] R.sub.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0258] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0259] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O)m-R.sup.14, or
--(R.sup.13O)m-CH(R.sup.15).sub.2;
[0260] m is an integer ranging from 1-1000;
[0261] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0262] R.sup.14 is H or C.sup.1-C.sup.8 alkyl;
[0263] each occurrence of R.sup.15 is independently H, COON,
--(CH.sub.2)n-N(R.sup.16).sub.2, --(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2)n-SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0264] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH;
[0265] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sup.3--C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
[0266] n is an integer ranging from 0 to 6.
[0267] In another aspect, the antibody of the antibody-drug
conjugate (ADC) of the invention specifically binds to a receptor
encoded by an ErbB2 gene.
[0268] In another aspect, the antibody of the antibody-drug
conjugate is a humanized antibody selected from huMAb4D5-1,
huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,
huMAb4D5-7 and huMAb4D5-8 (Trastuzumab).
[0269] In another aspect, the invention includes an article of
manufacture comprising an antibody-drug conjugate compound of the
invention; a container; and a package insert or label indicating
that the compound can be used to treat cancer characterized by the
overexpression of an ErbB2 receptor.
[0270] In another aspect, the invention includes a method for the
treatment of cancer in a mammal, wherein the cancer is
characterized by the overexpression of an ErbB2 receptor and does
not respond, or responds poorly, to treatment with an anti-ErbB2
antibody, comprising administering to the mammal a therapeutically
effective amount of an antibody-drug conjugate compound of the
invention.
[0271] In another aspect, a substantial amount of the drug moiety
is not cleaved from the antibody until the antibody-drug conjugate
compound enters a cell with a cell-surface receptor specific for
the antibody of the antibody-drug conjugate, and the drug moiety is
cleaved from the antibody when the antibody-drug conjugate does
enter the cell.
[0272] In another aspect, the bioavailability of the antibody-drug
conjugate compound or an intracellular metabolite of the compound
in a mammal is improved when compared to a drug compound comprising
the drug moiety of the antibody-drug conjugate compound, or when
compared to an analog of the compound not having the drug
moiety.
[0273] In another aspect, the drug moiety is intracellularly
cleaved in a mammal from the antibody of the compound, or an
intracellular metabolite of the compound.
[0274] In another aspect, the invention includes a pharmaceutical
composition comprising an effective amount of the antibody-drug
conjugate compound of the invention, or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable diluent,
carrier or excipient. The composition may further comprise a
therapeutically effective amount of chemotherapeutic agent such as
a tubulin-forming inhibitor, a topoisomerase inhibitor, and a DNA
binder.
[0275] In another aspect, the invention includes a method for
killing or inhibiting the proliferation of tumor cells or cancer
cells comprising treating tumor cells or cancer cells with an
amount of the antibody-drug conjugate compound of the invention, or
a pharmaceutically acceptable salt or solvate thereof, being
effective to kill or inhibit the proliferation of the tumor cells
or cancer cells.
[0276] In another aspect, the invention includes a method of
inhibiting cellular proliferation comprising exposing mammalian
cells in a cell culture medium to an antibody drug conjugate
compound of the invention, wherein the antibody drug conjugate
compound enters the cells and the drug is cleaved from the
remainder of the antibody drug conjugate compound; whereby
proliferation of the cells is inhibited.
[0277] In another aspect, the invention includes a method of
treating cancer comprising administering to a patient a formulation
of an antibody-drug conjugate compound of the invention and a
pharmaceutically acceptable diluent, carrier or excipient.
[0278] In another aspect, the invention includes an assay for
detecting cancer cells comprising: [0279] (a) exposing cells to an
antibody-drug conjugate compound of the invention; and [0280] (b)
determining the extent of binding of the antibody-drug conjugate
compound to the cells.
[0281] The invention will best be understood by reference to the
following detailed description of the exemplary embodiments, taken
in conjunction with the accompanying drawings, figures, and
schemes. The discussion below is descriptive, illustrative and
exemplary and is not to be taken as limiting the scope defined by
any appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0282] FIG. 1 shows an in vivo, single dose, efficacy assay of
cAC10-mcMMAF in subcutaneous Karpas-299 ALCL xenografts.
[0283] FIG. 2 shows an in vivo, single dose, efficacy assay of
cAC10-mcMMAF in subcutaneous L540cy. For this study there were 4
mice in the untreated group and 10 in each of the treatment
groups.
[0284] FIGS. 3a and 3b show in vivo efficacy of cBR96-mcMMAF in
subcutaneous L2987. The filed triangles in FIG. 3a and arrows in
FIG. 3b indicate the days of therapy.
[0285] FIGS. 4a and 4b show in vitro activity of
cAC10-antibody-drug conjugates against CD30.sup.+ cell lines.
[0286] FIGS. 5a and 5b show in vitro activity of
cBR96-antibody-drug conjugates against Le.sup.y+ cell lines.
[0287] FIGS. 6a and 6b show in vitro activity of c1F6-antibody-drug
conjugates against CD70.sup.+ renal cell carcinoma cell lines.
[0288] FIG. 7 shows an in vitro, cell proliferation assay with
SK-BR-3 cells treated with antibody drug conjugates (ADC): - -
Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -o- Trastuzumab-MC-MMAF,
4.1 MMAF/Ab, and -.DELTA.- Trastuzumab-MC-MMAF, 4.8 MMAF/Ab,
measured in Relative Fluorescence Units (RLU) versus .mu.g/ml
concentration of ADC. - - Trastuzumab where H is linked via a
cysteine [cys].
[0289] FIG. 8 shows an in vitro, cell proliferation assay with
BT-474 cells treated with ADC: - - Trastuzumab-MC-vc-PAB-MMAF, 3.8
MMAF/Ab, -o- Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -.DELTA.-
Trastuzumab-MC-MMAF, 4.8 MMAF/Ab.
[0290] FIG. 9 shows an in vitro, cell proliferation assay with
MCF-7 cells treated with ADC: - - Trastuzumab-MC-vc-PAB-MMAF, 3.8
MMAF/Ab, -o- Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab, and
-.DELTA.- Trastuzumab-MC-MMAF, 4.1 MMAF/Ab.
[0291] FIG. 10 shows an in vitro, cell proliferation assay with
MDA-MB-468 cells treated with ADC: - - Trastuzumab-MC-vc-PAB-MMAE,
4.1 MMAE/Ab, -o-Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab, and
-.DELTA.- Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.
[0292] FIG. 11 shows a plasma concentration clearance study after
administration of H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to
Sprague-Dawley rats: The administered dose was 2 mg of ADC per kg
of rat. Concentrations of total antibody and ADC were measured over
time. (H=Trastuzumab).
[0293] FIG. 12 shows a plasma concentration clearance study after
administration of H-MC-vc-MMAE to Cynomolgus monkeys at different
doses: 0.5, 1.5, 2.5, and 3.0 mg/kg administered at day 1 and day
21. Concentrations of total antibody and ADC were measured over
time. (H=Trastuzumab).
[0294] FIG. 13 shows the mean tumor volume change over time in
athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed
on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250
.mu.g/m.sup.2) and Trastuzumab-MC-vc-PAB-MMAF (555 .mu.g/m.sup.2).
(H=Trastuzumab).
[0295] FIG. 14 shows the mean tumor volume change over time in
athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed
on Day 0 with 10 mg/kg (660 .mu.g/m.sup.2) of Trastuzumab-MC-MMAE
and 1250 .mu.g/m.sup.2 Trastuzumab-MC-vc-PAB-MMAE.
[0296] FIG. 15 shows the mean tumor volume change over time in
athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed
on Day 0 with Vehicle and 650 .mu.g/m.sup.2
trastuzumab-MC-MMAF.
[0297] FIG. 16 shows the mean tumor volume change over time in
athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed
on Day 0 with Vehicle and 350 .mu.g/m.sup.2 of four
trastuzumab-MC-MMAF conjugates where the MMAF/trastuzumab (H) ratio
is 2, 4, 5.9 and 6.
[0298] FIG. 17 shows the Group mean change, with error bars, in
animal (rat) body weights (Mean.+-.SD) after administration of
Vehicle, trastuzumab-MC-val-cit-MMAF,
trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and
trastuzumab-MC-val-cit-PAB-MMAF.
[0299] FIG. 18 shows the Group mean change in animal (rat) body
weights (Mean.+-.SD) after administration of 9.94 mg/kg
H-MC-vc-MMAF, 24.90 mg/kg H-MC-vc-MMAF, 10.69 mg/kg
H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kg H-MC(Me)-vc-PAB-MMAF, 10.17 mg/kg
H-MC-MMAF, 25.50 mg/kg H-MC-MMAF, and 21.85 mg/kg H-MC-vc-PAB-MMAF.
H=trastuzumab. The MC linker is attached via a cysteine of
trastuzumab for each conjugate.
[0300] FIG. 19 shows the Group mean change, with error bars, in
Sprague Dawley rat body weights (Mean.+-.SD) after administration
of trastuzumab (H)-MC-MMAF at doses of 2105, 3158, and 4210
.mu.g/m.sup.2. The MC linker is attached via a cysteine of
trastuzumab for each conjugate.
[0301] FIG. 20 shows examples of compounds with a non
self-immolative Spacer unit.
[0302] FIG. 21 shows a scheme of a possible mechanism of Drug
release from a PAB group which is attached directly to -D via a
carbamate or carbonate group.
[0303] FIG. 22 shows a scheme of a possible mechanism of Drug
release from a PAB group which is attached directly to -D via an
ether or amine linkage.
[0304] FIG. 23 shows an example of a branched spacer unit,
bis(hydroxymethyl)styrene (BHMS) unit, which can be used to
incorporate and release multiple drug.
[0305] FIG. 24 shows a scheme of the CellTiter-Glo.RTM. Assay.
[0306] FIG. 25 shows the synthesis of an N-terminal tripeptide unit
F which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
[0307] FIG. 26 shows the synthesis of an N-terminal tripeptide unit
F which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
[0308] FIG. 27 shows the synthesis of an N-terminal tripeptide unit
F which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
[0309] FIG. 28 shows the synthesis of useful linkers.
[0310] FIG. 29 shows the synthesis of useful linkers.
[0311] FIG. 30 shows a general synthesis of an illustrative Linker
unit containing a maleimide Stretcher group and optionally a
p-aminobenzyl ether self-immolative Spacer.
[0312] FIG. 31 shows the synthesis of a val-cit dipeptide Linker
having a maleimide Stretcher and optionally a p-aminobenzyl
self-immolative Spacer.
[0313] FIG. 32 shows the synthesis of a phe-lys(Mtr) dipeptide
Linker unit having a maleimide Stretcher unit and a p-aminobenzyl
self-immolative Spacer unit.
[0314] FIG. 33 shows the synthesis of a Drug-Linker Compound that
contains an amide or carbamate group, linking the Drug unit to the
Linker unit.
[0315] FIG. 34 shows illustrative methods useful for linking a Drug
to a Ligand to form a Drug-Linker Compound.
[0316] FIG. 35 shows the synthesis of a val-cit dipeptide linker
having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene
(BHMS) unit.
[0317] FIG. 36 shows methodology useful for making
Drug-Linker-Ligand conjugates having about 2 to about 4 drugs per
antibody.
[0318] FIG. 37 shows the synthesis of MC-MMAF via t-butyl
ester.
[0319] FIG. 38 shows the synthesis of MC-MMAF (11) via
dimethoxybenzyl ester.
[0320] FIG. 39 shows the synthesis of analog of mc-MMAF.
4. DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
4.1 Definitions and Abbreviations
[0321] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings:
[0322] When trade names are used herein, applicants intend to
independently include the trade name product formulation, the
generic drug, and the active pharmaceutical ingredient(s) of the
trade name product.
[0323] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments,
so long as they exhibit the desired biological activity. An
antibody is a protein generated by the immune system that is
capable of recognizing and binding to a specific antigen. Described
in terms of its structure, an antibody typically has a Y-shaped
protein consisting of four amino acid chains, two heavy and two
light. Each antibody has primarily two regions: a variable region
and a constant region. The variable region, located on the ends of
the arms of the Y, binds to and interacts with the target antigen.
This variable region includes a complementary determining region
(CDR) that recognizes and binds to a specific binding site on a
particular antigen. The constant region, located on the tail of the
Y, is recognized by and interacts with the immune system (Janeway,
C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th
Ed., Garland Publishing, New York). A target antigen generally has
numerous binding sites, also called epitopes, recognized by CDRs on
multiple antibodies. Each antibody that specifically binds to a
different epitope has a different structure. Thus, one antigen may
have more than one corresponding antibody.
[0324] The term "antibody" as used herein, also refers to a
full-length immunoglobulin molecule or an immunologically active
portion of a full-length immunoglobulin molecule, i.e., a molecule
that contains an antigen binding site that immunospecifically binds
an antigen of a target of interest or part thereof, such targets
including but not limited to, cancer cell or cells that produce
autoimmune antibodies associated with an autoimmune disease. The
immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE,
IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule. The immunoglobulins
can be derived from any species. In one aspect, however, the
immunoglobulin is of human, murine, or rabbit origin. In another
aspect, the antibodies are polyclonal, monoclonal, bispecific,
human, humanized or chimeric antibodies, single chain antibodies,
Fv, Fab fragments, F(ab') fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, CDR's, and epitope-binding fragments of any
of the above which immunospecifically bind to cancer cell antigens,
viral antigens or microbial antigens.
[0325] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.
(1975) Nature 256:495, or may be made by recombinant DNA methods
(see, U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may
also be isolated from phage antibody libraries using the techniques
described in Clackson et al. (1991) Nature, 352:624-628 and Marks
et al. (1991) J. Mol. Biol., 222:581-597, for example.
[0326] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl.
Acad. Sci. USA, 81:6851-6855).
[0327] Various methods have been employed to produce monoclonal
antibodies (MAbs). Hybridoma technology, which refers to a cloned
cell line that produces a single type of antibody, uses the cells
of various species, including mice (murine), hamsters, rats, and
humans. Another method to prepare MAbs uses genetic engineering
including recombinant DNA techniques. Monoclonal antibodies made
from these techniques include, among others, chimeric antibodies
and humanized antibodies. A chimeric antibody combines DNA encoding
regions from more than one type of species. For example, a chimeric
antibody may derive the variable region from a mouse and the
constant region from a human. A humanized antibody comes
predominantly from a human, even though it contains nonhuman
portions. Like a chimeric antibody, a humanized antibody may
contain a completely human constant region. But unlike a chimeric
antibody, the variable region may be partially derived from a
human. The nonhuman, synthetic portions of a humanized antibody
often come from CDRs in murine antibodies. In any event, these
regions are crucial to allow the antibody to recognize and bind to
a specific antigen.
[0328] As noted, murine antibodies can be used. While useful for
diagnostics and short-term therapies, murine antibodies cannot be
administered to people long-term without increasing the risk of a
deleterious immunogenic response. This response, called Human
Anti-Mouse Antibody (HAMA), occurs when a human immune system
recognizes the murine antibody as foreign and attacks it. A HAMA
response can cause toxic shock or even death.
[0329] Chimeric and humanized antibodies reduce the likelihood of a
HAMA response by minimizing the nonhuman portions of administered
antibodies. Furthermore, chimeric and humanized antibodies have the
additional benefit of activating secondary human immune responses,
such as antibody dependent cellular cytotoxicity.
[0330] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding or variable
region thereof. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragment(s).
[0331] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The
constant domains may be native sequence constant domains (e.g.,
human native sequence constant domains) or amino acid sequence
variant thereof.
[0332] The intact antibody may have one or more "effector
functions" which refer to those biological activities attributable
to the Fc region (a native sequence Fc region or amino acid
sequence variant Fc region) of an antibody. Examples of antibody
effector functions include C1q binding; complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor; BCR), etc.
[0333] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes." There are five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0334] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to human HER2 protein described, for example, in
Semba et al., Proc. Natl. Acad. Sci. USA, 82:6497-6501 (1985) and
Yamamoto et al., (1986) Nature, 319:230-234 (Genebank accession
number X03363). The term "erbB2" refers to the gene encoding human
ErbB2 and "neu" refers to the gene encoding rat p185neu. Preferred
ErbB2 is native sequence human ErbB2.
[0335] Antibodies to ErbB receptors are available commercially from
a number of sources, including, for example, Santa Cruz
Biotechnology, Inc., California, USA.
[0336] By "ErbB ligand" is meant a polypeptide which binds to
and/or activates an ErbB receptor. The ErbB ligand may be a native
sequence human ErbB ligand such as epidermal growth factor (EGF)
(Savage et al. (1972) J. Biol. Chem., 247:7612-7621); transforming
growth factor alpha (TGF-.alpha.) (Marquardt et al. (1984) Science
223:1079-1082); amphiregulin also known as schwanoma or
keratinocyte autocrine growth factor (Shoyab et al. (1989) Science
243:1074-1076; Kimura et al., Nature, 348:257-260 (1990); and Cook
et al., Mol. Cell. Biol., 11:2547-2557 (1991)); betacellulin (Shing
et al., Science, 259:1604-1607 (1993); and Sasada et al., Biochem.
Biophys. Res. Commun., 190:1173 (1993)); heparin-binding epidermal
growth factor (HB-EGF) (Higashiyama et al., Science, 251:936-939
(1991)); epiregulin (Toyoda et al., J. Biol. Chem., 270:7495-7500
(1995); and Komurasaki et al., Oncogene, 15:2841-2848 (1997)); a
heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al.,
Nature, 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al.,
Proc. Natl. Acad. Sci., 94:9562-9567 (1997)); neuregulin-4 (NRG-4)
(Harari et al., Oncogene, 18:2681-89 (1999)) or cripto (CR-1)
(Kannan et al., J. Biol. Chem., 272(6):3330-3335 (1997)). ErbB
ligands which bind EGFR include EGF, TGF-.alpha., amphiregulin,
betacellulin, HB-EGF and epiregulin. ErbB ligands which bind ErbB3
include heregulins. ErbB ligands capable of binding ErbB4 include
betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and
heregulins. The ErbB ligand may also be a synthetic ErbB ligand.
The synthetic ligand may be specific for a particular ErbB
receptor, or may recognize particular ErbB receptor complexes. An
example of a synthetic ligand is the synthetic heregulin/EGF
chimera biregulin (see, for example, Jones et al., (1999) FEBS
Letters, 447:227-231, which is incorporated by reference).
[0337] "Heregulin" (HRG) refers to a polypeptide encoded by the
heregulin gene product as disclosed in U.S. Pat. No. 5,641,869 or
Marchionni et al., Nature, 362:312-318 (1993). Examples of
heregulins include heregulin-.alpha., heregulin-.beta.1,
heregulin-.beta.2 and heregulin-.beta.3 (Holmes et al., Science,
256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu
differentiation factor (NDF) (Peles et al., Cell 69: 205-216
(1992)); acetylcholine receptor-inducing activity (ARIA) (Falls et
al. (1993) Cell 72:801-815); glial growth factors (GGFs)
(Marchionni et al., Nature, 362:312-318 (1993)); sensory and motor
neuron derived factor (SMDF) (Ho et al., J. Biol. Chem.,
270:14523-14532 (1995)); .gamma.-heregulin (Schaefer et al.,
Oncogene, 15:1385-1394 (1997)). The term includes biologically
active fragments and/or amino acid sequence variants of a native
sequence HRG polypeptide, such as an EGF-like domain fragment
thereof (e.g., HRG.beta.1177-244).
[0338] "ErbB hetero-oligomer" is a noncovalently associated
oligomer comprising at least two different ErbB receptors. An "ErbB
dimer" is a noncovalently associated oligomer that comprises two
different ErbB receptors. Such complexes may form when a cell
expressing two or more ErbB receptors is exposed to an ErbB ligand.
ErbB oligomers, such as ErbB dimers, can be isolated by
immunoprecipitation and analyzed by SDS-PAGE as described in
Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), for
example. Examples of such ErbB hetero-oligomers include EGFR-ErbB2
(also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and
ErbB3-ErbB4 (HER3/HER4) complexes. Moreover, the ErbB
hetero-oligomer may comprise two or more ErbB2 receptors combined
with a different ErbB receptor, such as ErbB3, ErbB4 or EGFR
(ErbB1). Other proteins, such as a cytokine receptor subunit (e.g.,
gp130) may be included in the hetero-oligomer.
[0339] A "native sequence" polypeptide is one which has the same
amino acid sequence as a polypeptide, e.g., tumor-associated
antigen receptor, derived from nature. Such native sequence
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. Thus, a native sequence polypeptide
can have the amino acid sequence of naturally-occurring human
polypeptide, murine polypeptide, or polypeptide from any other
mammalian species.
[0340] The term "amino acid sequence variant" refers to
polypeptides having amino acid sequences that differ to some extent
from a native sequence polypeptide. Ordinarily, amino acid sequence
variants will possess at least about 70% homology with at least one
receptor binding domain of a native ligand, or with at least one
ligand binding domain of a native receptor, such as a
tumor-associated antigen, and preferably, they will be at least
about 80%, more preferably, at least about 90% homologous with such
receptor or ligand binding domains. The amino acid sequence
variants possess substitutions, deletions, and/or insertions at
certain positions within the amino acid sequence of the native
amino acid sequence.
[0341] "Sequence identity" is defined as the percentage of residues
in the amino acid sequence variant that are identical after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Methods and computer
programs for the alignment are well known in the art. One such
computer program is "Align 2," authored by Genentech, Inc., which
was filed with user documentation in the United States Copyright
Office, Washington, D.C. 20559, on Dec. 10, 1991.
[0342] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, (1991) Annu. Rev.
Immunol, 9:457-92. To assess ADCC activity of a molecule of
interest, an in vitro ADCC assay, such as that described in U.S.
Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector
cells for such assays include peripheral blood mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al., Prco. Natl. Acad. Set USA, 95:652-656 (1998).
[0343] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(See review M. in Daeron, Annu. Rev. Immunol., 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol.,
9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and
de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs,
including those to be identified in the future, are encompassed by
the term "FcR" herein. The term also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus. (Guyer et al., J. Immunol., 117:587 (1976) and
Kim et al., J. Immunol., 24:249 (1994)).
[0344] "Complement dependent cytotoxicity" or "CDC" refers to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (Clq) to a
molecule (e.g., an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be
performed.
[0345] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al. (1991) Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md.). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody dependent cellular cytotoxicity (ADCC).
[0346] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al. supra) and/or those
residues from a "hypervariable loop" (e.g., residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0347] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0348] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six hypervariable regions confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three
hypervariable regions specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
[0349] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0350] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0351] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Preferably, the Fv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0352] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a variable
heavy domain (VH) connected to a variable light domain (VL) in the
same polypeptide chain (VH-VL). By using a linker that is too short
to allow pairing between the two domains on the same chain, the
domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are
described more fully in, for example, EP 404,097; WO 93/11161; and
Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448.
[0353] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al. (1986)
Nature, 321:522-525; Riechmann et al. (1988) Nature 332:323-329;
and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.
[0354] Humanized anti-ErbB2 antibodies include huMAb4D5-1,
huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,
huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN.RTM.) as described in Table 3
of U.S. Pat. No. 5,821,337 expressly incorporated herein by
reference; humanized 520C9 (WO 93/21319) and humanized 2C4
antibodies as described herein below.
[0355] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0356] An antibody "which binds" an antigen of interest is one
capable of binding that antigen with sufficient affinity such that
the antibody is useful in targeting a cell expressing the
antigen.
[0357] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is a tumor cell, e.g.,
a breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic or bladder cell. Various methods
are available for evaluating the cellular events associated with
apoptosis. For example, phosphatidyl serine (PS) translocation can
be measured by annexin binding; DNA fragmentation can be evaluated
through DNA laddering; and nuclear/chromatin condensation along
with DNA fragmentation can be evaluated by any increase in
hypodiploid cells.
[0358] A "disorder" is any condition that would benefit from
treatment of the present invention. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question. Non-limiting
examples of disorders to be treated herein include benign and
malignant tumors; leukemia and lymphoid malignancies, in particular
breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic, prostate or bladder cancer;
neuronal, glial, astrocytal, hypothalamic and other glandular,
macrophagal, epithelial, stromal and blastocoelic disorders; and
inflammatory, angiogenic and immunologic disorders.
[0359] The term "therapeutically effective amount" refers to an
amount of a drug effective to treat a disease or disorder in a
mammal. In the case of cancer, the therapeutically effective amount
of the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the cancer. To the extent the
drug may prevent growth and/or kill existing cancer cells, it may
be cytostatic and/or cytotoxic. For cancer therapy, efficacy can,
for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
[0360] The term "substantial amount" refers to a majority, i.e.
>50% of a population, of a collection or a sample.
[0361] The term "intracellular metabolite" refers to a compound
resulting from a metabolic process or reaction inside a cell on an
antibody drug conjugate (ADC). The metabolic process or reaction
may be an enzymatic process such as proteolytic cleavage of a
peptide linker of the ADC, or hydrolysis of a functional group such
as a hydrazone, ester, or amide. Intracellular metabolites include,
but are not limited to, antibodies and free drug which have
undergone intracellular cleavage after entry, diffusion, uptake or
transport into a cell.
[0362] The terms "intracellularly cleaved" and "intracellular
cleavage" refer to a metabolic process or reaction inside a cell on
an Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate, an
antibody drug conjugate (ADC) or the like whereby the covalent
attachment, e.g., the linker, between the drug moiety (D) and the
antibody (Ab) is broken, resulting in the free drug dissociated
from the antibody inside the cell. The cleaved moieties of the
Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate or ADC are
thus intracellular metabolites.
[0363] The term "bioavailability" refers to the systemic
availability (i.e., blood/plasma levels) of a given amount of drug
administered to a patient. Bioavailability is an absolute term that
indicates measurement of both the time (rate) and total amount
(extent) of drug that reaches the general circulation from an
administered dosage form.
[0364] The term "cytotoxic activity" refers to a cell-killing,
cytostatic or anti-proliferation effect of an antibody drug
conjugate compound or an intracellular metabolite of an antibody
drug conjugate compound. Cytotoxic activity may be expressed as the
IC.sub.50 value which is the concentration (molar or mass) per unit
volume at which half the cells survive.
[0365] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells. Examples of cancer include, but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer including gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer.
[0366] An "ErbB2-expressing cancer" is one which produces
sufficient levels of ErbB2 at the surface of cells thereof, such
that an anti-ErbB2 antibody can bind thereto and have a therapeutic
effect with respect to the cancer.
[0367] A cancer "characterized by excessive activation" of an ErbB2
receptor is one in which the extent of ErbB2 receptor activation in
cancer cells significantly exceeds the level of activation of that
receptor in non-cancerous cells of the same tissue type. Such
excessive activation may result from overexpression of the ErbB2
receptor and/or greater than normal levels of an ErbB2 ligand
available for activating the ErbB2 receptor in the cancer cells.
Such excessive activation may cause and/or be caused by the
malignant state of a cancer cell. In some embodiments, the cancer
will be subjected to a diagnostic or prognostic assay to determine
whether amplification and/or overexpression of an ErbB2 receptor is
occurring which results in such excessive activation of the ErbB2
receptor. Alternatively, or additionally, the cancer may be
subjected to a diagnostic or prognostic assay to determine whether
amplification and/or overexpression an ErbB2 ligand is occurring in
the cancer which attributes to excessive activation of the
receptor. In a subset of such cancers, excessive activation of the
receptor may result from an autocrine stimulatory pathway.
[0368] A cancer which "overexpresses" an ErbB2 receptor is one
which has significantly higher levels of an ErbB2 receptor at the
cell surface thereof, compared to a noncancerous cell of the same
tissue type. Such overexpression may be caused by gene
amplification or by increased transcription or translation. ErbB2
receptor overexpression may be determined in a diagnostic or
prognostic assay by evaluating increased levels of the ErbB2
protein present on the surface of a cell (e.g., via an
immunohistochemistry assay; IHC). Alternatively, or additionally,
one may measure levels of ErbB2-encoding nucleic acid in the cell,
e.g., via fluorescent in situ hybridization (FISH; see WO
98/45479), southern blotting, or polymerase chain reaction (PCR)
techniques, such as real time quantitative PCR (RT-PCR).
Overexpression of the ErbB2 ligand, may be determined
diagnostically by evaluating levels of the ligand (or nucleic acid
encoding it) in the patient, e.g., in a tumor biopsy or by various
diagnostic assays such as the IHC, FISH, southern blotting, PCR or
in vivo assays described above. One may also study ErbB2 receptor
overexpression by measuring shed antigen (e.g., ErbB2 extracellular
domain) in a biological fluid such as serum (see, e.g., U.S. Pat.
No. 4,933,294; WO 91/05264; U.S. Pat. No. 5,401,638; and Sias et
al., (1990) J. Immunol. Methods, 132: 73-80). Aside from the above
assays, various other in vivo assays are available to the skilled
practitioner. For example, one may expose cells within the body of
the patient to an antibody which is optionally labeled with a
detectable label, e.g., a radioactive isotope, and binding of the
antibody to cells in the patient can be evaluated, e.g., by
external scanning for radioactivity or by analyzing a biopsy taken
from a patient previously exposed to the antibody.
[0369] The tumors overexpressing HER2 are rated by
immunohistochemical scores corresponding to the number of copies of
HER2 molecules expressed per cell, and can been determined
biochemically: 0=0-10,000 copies/cell, 1+=at least about 200,000
copies/cell, 2+=at least about 500,000 copies/cell, 3+=about
1-2.times.10.sup.6 copies/cell. Overexpression of HER2 at the 3+
level, which leads to ligand-independent activation of the tyrosine
kinase (Hudziak et al., (1987) Proc. Natl. Acad. Sci. USA,
84:7159-7163), occurs in approximately 30% of breast cancers, and
in these patients, relapse-free survival and overall survival are
diminished (Slamon et al., (1989) Science, 244:707-712; Slamon et
al., (1987) Science, 235:177-182).
[0370] Conversely, a cancer which is "not characterized by
overexpression of the ErbB2 receptor" is one which, in a diagnostic
assay, does not express higher than normal levels of ErbB2 receptor
compared to a noncancerous cell of the same tissue type.
[0371] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., .sup.211At, .sup.131I, .sup.125I,
.sup.90Y, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P,
.sup.60C, and radioactive isotopes of Lu), chemotherapeutic agents,
and toxins such as small molecule toxins or enzymatically active
toxins of bacterial, fungal, plant or animal origin, including
synthetic analogs and derivatives thereof. In one aspect, the term
is not intended to include radioactive isotopes.
[0372] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; TLK 286 (TELCYTA.TM.); acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; bisphosphonates, such as
clodronate; antibiotics such as the enediyne antibiotics (e.g.,
calicheamicin, especially calicheamicin gamma1I and calicheamicin
omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186
(1994)) and anthracyclines such as annamycin, AD 32, alcarubicin,
daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100,
idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A,
an esperamicin, neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores, aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal
doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, and zorubicin; folic acid analogues such as denopterin,
pteropterin, and trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs
such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, and
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, and testolactone;
anti-adrenals such as aminoglutethimide, mitotane, and trilostane;
folic acid replenisher such as folinic acid (leucovorin);
aceglatone; anti-folate anti-neoplastic agents such as ALIMTA.RTM.,
LY231514 pemetrexed, dihydrofolate reductase inhibitors such as
methotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and
its prodrugs such as UFT, S-1 and capecitabine, and thymidylate
synthase inhibitors and glycinamide ribonucleotide
formyltransferase inhibitors such as raltitrexed (TOMUDEX.RTM.,
TDX); inhibitors of dihydropyrimidine dehydrogenase such as
eniluracil; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate; an
epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins;
mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;
phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g.,
TAXOL.RTM. paclitaxel (Bristol-Myers Squibb Oncology, Princeton,
N.J.), ABRAXANE.TM. Cremophor-free, albumin-engineered nanoparticle
formulation of paclitaxel (American Pharmaceutical Partners,
Schaumberg, Ill.), and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc
Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR.RTM.);
6-thioguanine; mercaptopurine; platinum; platinum analogs or
platinum-based analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine (VELBAN.RTM.); etoposide (VP-16);
ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.); vinca
alkaloid; vinorelbine (NAVELBINE.RTM.); novantrone; edatrexate;
daunomycin; aminopterin; xeloda; ibandronate; topoisomerase
inhibitor RFS 2000; difluorometihylornithine (DMFO); retinoids such
as retinoic acid; pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone, and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and
leucovorin.
[0373] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON.RTM.
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras,
and epidermal growth factor receptor (EGF-R); vaccines such as gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; PROLEUKIN.RTM.
rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; and pharmaceutically acceptable salts, acids or derivatives
of any of the above.
[0374] As used herein, the term "EGFR-targeted drug" refers to a
therapeutic agent that binds to EGFR and, optionally, inhibits EGFR
activation. Examples of such agents include antibodies and small
molecules that bind to EGFR. Examples of antibodies which bind to
EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),
MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat.
No. 4,943,533, Mendelsohn et al.) and variants thereof, such as
chimerized 225 (C225 or Cetuximab; ERBITUX.RTM.) and reshaped human
225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies
that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized
and chimeric antibodies that bind EGFR as described in U.S. Pat.
No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF
(see WO 98/50433, Abgenix). The anti-EGFR antibody may be
conjugated with a cyotoxic agent, thus generating an
immunoconjugate (see, e.g., EP 659,439A2, Merck patent GmbH).
Examples of small molecules that bind to EGFR include ZD1839 or
Gefitinib (IRESSA.TM.; Astra Zeneca), Erlotinib HCl (CP-358774,
TARCEVA.TM.; Genentech/OSI) and AG1478, AG1571 (SU 5271;
Sugen).
[0375] A "tyrosine kinase inhibitor" is a molecule which inhibits
to some extent tyrosine kinase activity of a tyrosine kinase such
as an ErbB receptor. Examples of such inhibitors include the
EGFR-targeted drugs noted in the preceding paragraph as well as
quinazolines such as PD 153035, 4-(3-chloroanilino) quinazoline,
pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as
CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lambert);
antisense molecules (e.g., those that bind to ErbB-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate
(Gleevac; Novartis); PM 166 (Novartis); GW2016 (Glaxo SmithKline);
CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474
(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone);
or as described in any of the following patent publications: U.S.
Pat. No. 5,804,396; WO 99/09016 (American Cyanamid); WO 98/43960
(American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378
(Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347
(Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO
96/33980 (Zeneca).
[0376] An "anti-angiogenic agent" refers to a compound which
blocks, or interferes with to some degree, the development of blood
vessels. The anti-angiogenic factor may, for instance, be a small
molecule or antibody that binds to a growth factor or growth factor
receptor involved in promoting angiogenesis. In one embodiment, the
anti-angiogenic factor is an antibody that binds to Vascular
Endothelial Growth Factor (VEGF).
[0377] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the team
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0378] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically or hydrolytically activated
or converted into the more active parent form. See, e.g., Wilman,
"Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions,
14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al.,
"Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed
Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0379] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as including the anti-CD30, CD40, CD70 or
Lewis Y antibodies and, optionally, a chemotherapeutic agent) to a
mammal. The components of the liposome are commonly arranged in a
bilayer formation, similar to the lipid arrangement of biological
membranes.
[0380] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0381] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the faun or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the antibody where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0382] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0383] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking can be accomplished
by ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers can be
used in accordance with conventional practice.
[0384] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0385] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and
ankylosing spondylitis), psoriasis, dermatitis including atopic
dermatitis; chronic idiopathic urticaria, including chronic
autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal
necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease (IBD) (Crohn's disease,
ulcerative colitis), and IBD with co-segregate of pyoderma
gangrenosum, erythema nodosum, primary sclerosing cholangitis,
and/or episcleritis), respiratory distress syndrome, including
adult respiratory distress syndrome (ARDS), meningitis,
IgE-mediated diseases such as anaphylaxis and allergic rhinitis,
encephalitis such as Rasmussen's encephalitis, uveitis, colitis
such as microscopic colitis and collagenous colitis,
glomerulonephritis (GN) such as membranous GN, idiopathic
membranous GN, membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
eczema, asthma, conditions involving infiltration of T cells and
chronic inflammatory responses, atherosclerosis, autoimmune
myocarditis, leukocyte adhesion deficiency, systemic lupus
erythematosus (SLE) such as cutaneous SLE, lupus (including
nephritis, cerebritis, pediatric, non-renal, discoid, alopecia),
juvenile onset diabetes, multiple sclerosis (MS) such as
spino-optical MS, allergic encephalomyelitis, immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including Wegener's granulomatosis, agranulocytosis,
vasculitis (including Large Vessel vasculitis (including
Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis),
Medium Vessel vasculitis (including Kawasaki's Disease and
Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated
vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)),
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
immune hemolytic anemia including autoimmune hemolytic anemia
(AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome,
myasthenia gravis, antigen-antibody complex mediated diseases,
anti-glomerular basement membrane disease, anti-phospholipid
antibody syndrome, allergic neuritis, Bechet disease, Castleman's
syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic
Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson
syndrome, solid organ transplant rejection (including pretreatment
for high panel reactive antibody titers, IgA deposit in tissues,
and rejection arising from renal transplantation, liver
transplantation, intestinal transplantation, cardiac
transplantation, etc.), graft versus host disease (GVHD),
pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and
pemphigus mucus-membrane pemphigoid), autoimmune
polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune
complex nephritis, IgM polyneuropathies or IgM mediated neuropathy,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), thrombocytopenia (as developed by
myocardial infarction patients, for example), including autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimmune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM),
including pediatric IDDM, and Sheehan's syndrome; autoimmune
hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis
obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome,
Berger's Disease (IgA nephropathy), primary biliary cirrhosis,
celiac sprue (gluten enteropathy), refractory sprue with
co-segregate dermatitis herpetiformis, cryoglobulinemia,
amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease),
coronary artery disease, autoimmune inner ear disease (AIED),
autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS),
polychondritis such as refractory polychondritis, pulmonary
alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,
monoclonal gammopathy of uncertain/unknown significance (MGUS),
peripheral neuropathy, paraneoplastic syndrome, channelopathies
such as epilepsy, migraine, arrhythmia, muscular disorders,
deafness, blindness, periodic paralysis, and channelopathies of the
CNS; autism, inflammatory myopathy, and focal segmental
glomerulosclerosis (FSGS).
[0386] "Alkyl" is C.sub.1-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms. Examples are methyl
(Me, --CH.sub.3), ethyl (Et, --CH.sub.2CH.sub.3), 1-propyl (n-Pr,
n-propyl, --CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (i-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3.
[0387] "Alkenyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp.sup.2 double bond.
Examples include, but are not limited to: ethylene or vinyl
(--CH.dbd.CH.sub.2), allyl (--CH.sub.2CH.dbd.CH.sub.2),
cyclopentenyl (--C.sub.5H.sub.7), and 5-hexenyl (--CH.sub.2
CH.sub.2CH.sub.2CH.sub.2CH.dbd.CH.sub.2).
[0388] "Alkynyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp triple bond. Examples
include, but are not limited to: acetylenic (--C.ident.CH) and
propargyl (--CH.sub.2C.ident.CH).
[0389] "Alkylene" refers to a saturated, branched or straight chain
or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkane. Typical alkylene radicals include, but are not limited to:
methylene (--CH.sub.2--) 1,2-ethyl (--CH.sub.2CH.sub.2--),
1,3-propyl (--CH.sub.2CH.sub.2CH.sub.2--), 1,4-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like.
[0390] "Alkenylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkene. Typical alkenylene radicals include, but are not
limited to: 1,2-ethylene (--CH.dbd.CH--).
[0391] "Alkynylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkyne. Typical alkynylene radicals include, but are not
limited to: acetylene propargyl (--CH.sub.2C.ident.C--), and
4-pentynyl (--CH.sub.2CH.sub.2CH.sub.2C.ident.CH--).
[0392] "Aryl" means a monovalent aromatic hydrocarbon radical of
6-20 carbon atoms derived by the removal of one hydrogen atom from
a single carbon atom of a parent aromatic ring system. Some aryl
groups are represented in the exemplary structures as "Ar". Typical
aryl groups include, but are not limited to, radicals derived from
benzene, substituted benzene, naphthalene, anthracene, biphenyl,
and the like.
[0393] "Arylalkyl" refers to an acyclic alkyl radical in which one
of the hydrogen atoms bonded to a carbon atom, typically a terminal
or sp.sup.3 carbon atom, is replaced with an aryl radical. Typical
arylalkyl groups include, but are not limited to, benzyl,
2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,
2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,
2-naphthophenylethan-1-yl and the like. The arylalkyl group
comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including
alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to
6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
[0394] "Heteroarylalkyl" refers to an acyclic alkyl radical in
which one of the hydrogen atoms bonded to a carbon atom, typically
a terminal or sp.sup.3 carbon atom, is replaced with a heteroaryl
radical. Typical heteroarylalkyl groups include, but are not
limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The
heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g., the
alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the
heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl
moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from
N, O, P, and S. The heteroaryl moiety of the heteroarylalkyl group
may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and S), for example: a
bicyclo[4,5], [5,5], [5,6], or [6,6] system.
[0395] "Substituted alkyl", "substituted aryl", and "substituted
arylalkyl" mean alkyl, aryl, and arylalkyl respectively, in which
one or more hydrogen atoms are each independently replaced with a
substituent. Typical substituents include, but are not limited to,
--X, --R, --O.sup.-, --OR, --SR, --S.sup.-, --NR.sub.2, --NR.sub.3,
.dbd.NR, --CX.sub.3, --CN, --OCN, --SCN, --N.dbd.C.dbd.O, --NCS,
--NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3, NC(.dbd.O)R,
--C(.dbd.O)R, --C(.dbd.O)NR.sub.2, --SO.sub.3.sup.-, --SO.sub.3H,
--S(.dbd.O).sub.2R, --OS(.dbd.O).sub.2OR, --S(.dbd.O).sub.2NR,
--S(.dbd.O)R, --OP(.dbd.O)(OR).sub.2, --P(.dbd.O)(OR).sub.2,
--PO.sub.3.sup.-, --PO.sub.3H.sub.2, --C(.dbd.O)R, --C(.dbd.O)X,
--C(.dbd.S)R, --CO.sub.2R, --CO.sub.2.sup.-, --C(.dbd.S)OR,
--C(.dbd.O)SR, --C(.dbd.S)SR, --C(.dbd.O)NR.sub.2,
--C(.dbd.S)NR.sub.2, --C(.dbd.NR)NR.sub.2, where each X is
independently a halogen: F, Cl, Br, or I; and each R is
independently --H, C.sub.2-C.sub.18 alkyl, C.sub.6-C.sub.20 aryl,
C.sub.3-C.sub.14 heterocycle, protecting group or prodrug moiety.
Alkylene, alkenylene, and alkynylene groups as described above may
also be similarly substituted.
[0396] "Heteroaryl" and "Heterocycle" refer to a ring system in
which one or more ring atoms is a heteroatom, e.g., nitrogen,
oxygen, and sulfur. The heterocycle radical comprises 1 to 20
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. A
heterocycle may be a monocycle having 3 to 7 ring members (2 to 6
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S)
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and 5), for example: a
bicyclo[4,5], [5,5], [5,6], or [6,6] system.
[0397] Heterocycles are described in Paquette, Leo A.; "Principles
of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968),
particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of
Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,
19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
[0398] Examples of heterocycles include by way of example and not
limitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl),
thiazolyl, tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,
pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,
indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl,
pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,
tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl.
[0399] By way of example and not limitation, carbon bonded
heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine,
position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,
or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4
of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more
typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,
4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,
5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,
5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or
5-thiazolyl.
[0400] By way of example and not limitation, nitrogen bonded
heterocycles are bonded at position 1 of an aziridine, azetidine,
pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,
imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,
2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole,
indoline, 1H-indazole, position 2 of a isoindole, or isoindoline,
position 4 of a morpholine, and position 9 of a carbazole, or
.beta.-carboline. Still more typically, nitrogen bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0401] "Carbocycle" means a saturated or unsaturated ring having 3
to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a
bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still more
typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring
atoms, e.g., arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6]
system, or 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6]
system. Examples of monocyclic carbocycles include cyclopropyl,
cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and
cyclooctyl.
[0402] "Linker", "Linker Unit", or "link" means a chemical moiety
comprising a covalent bond or a chain of atoms that covalently
attaches an antibody to a drug moiety. In various embodiments, a
linker is specified as LU. Linkers include a divalent radical such
as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:
--(CR.sub.2).sub.nO(CR.sub.2).sub.n--, repeating units of alkyloxy
(e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.,
polyethyleneamino, Jeffamine.TM.); and diacid ester and amides
including succinate, succinamide, diglycolate, malonate, and
caproamide.
[0403] The teen "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0404] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0405] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties,
e.g., melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[0406] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0407] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds
(1994) John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and L, or R and S, are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and l or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or l meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these stereoisomers are identical except that
they are mirror images of one another. A specific stereoisomer may
also be referred to as an enantiomer, and a mixture of such isomers
is often called an enantiomeric mixture. A 50:50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species, devoid of optical activity.
[0408] Examples of a "patient" include, but are not limited to, a
human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog,
cat, bird and fowl. In an exemplary embodiment, the patient is a
human.
[0409] "Aryl" refers to a carbocyclic aromatic group. Examples of
aryl groups include, but are not limited to, phenyl, naphthyl and
anthracenyl. A carbocyclic aromatic group or a heterocyclic
aromatic group can be unsubstituted or substituted with one or more
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0410] The term "C.sub.1-C.sub.8 alkyl," as used herein refers to a
straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 8 carbon atoms. Representative "C.sub.1-C.sub.8
alkyl" groups include, but are not limited to, -methyl, -ethyl,
-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl,
-n-nonyl and -n-decyl; while branched C.sub.1-C.sub.8 alkyls
include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,
-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C.sub.1-C.sub.8
alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl,
-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,
1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl,
-2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl. methyl,
ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,
n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl,
3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl,
2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl,
2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl,
isoheptyl, n-octyl, and isooctyl. A C.sub.1-C.sub.8 alkyl group can
be unsubstituted or substituted with one or more groups including,
but not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --SO.sub.3R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; where each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0411] A "C.sub.3-C.sub.8 carbocycle" is a 3-, 4-, 5-, 6-, 7- or
8-membered saturated or unsaturated non-aromatic carbocyclic ring.
Representative C.sub.3-C.sub.8 carbocycles include, but are not
limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl,
-cyclopentadienyl, -cyclohexyl, -cyclohexenyl,
-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,
-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and
-cyclooctadienyl. A C.sub.3-C.sub.8 carbocycle group can be
unsubstituted or substituted with one or more groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; where each R' is independently selected from H,
--C.sub.1-C.sub.8 alkyl and aryl.
[0412] A "C.sub.3-C.sub.8 carbocyclo" refers to a C.sub.3-C.sub.8
carbocycle group defined above wherein one of the carbocycle
groups' hydrogen atoms is replaced with a bond.
[0413] A "C.sub.1-C.sub.10 alkylene" is a straight chain, saturated
hydrocarbon group of the formula --(CH.sub.2).sub.1-10--. Examples
of a C.sub.1-C.sub.10 alkylene include methylene, ethylene,
propylene, butylene, pentylene, hexylene, heptylene, ocytylene,
nonylene and decalene.
[0414] An "arylene" is an aryl group which has two covalent bonds
and can be in the ortho, meta, or para configurations as shown in
the following structures:
##STR00005##
in which the phenyl group can be unsubstituted or substituted with
up to four groups including, but not limited to, --C.sub.1-C.sub.8
alkyl, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0415] A "C.sub.3-C.sub.8 heterocycle" refers to an aromatic or
non-aromatic C.sub.3-C.sub.8 carbocycle in which one to four of the
ring carbon atoms are independently replaced with a heteroatom from
the group consisting of O, S and N. Representative examples of a
C.sub.3-C.sub.8 heterocycle include, but are not limited to,
benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl,
isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,
imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl,
pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl,
isoxazolyl and tetrazolyl. A C.sub.3-C.sub.8 heterocycle can be
unsubstituted or substituted with up to seven groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; wherein each R' is independently selected from H,
--C.sub.1-C.sub.8 alkyl and aryl.
[0416] "C.sub.3-C.sub.8 heterocyclo" refers to a C.sub.3-C.sub.8
heterocycle group defined above wherein one of the heterocycle
group's hydrogen atoms is replaced with a bond. A C.sub.3-C.sub.8
heterocyclo can be unsubstituted or substituted with up to six
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0417] An "Exemplary Compound" is a Drug Compound or a Drug-Linker
Compound.
[0418] An "Exemplary Conjugate" is a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate or a
Drug-Linker-Ligand Conjugate.
[0419] In some embodiments, the Exemplary Compounds and Exemplary
Conjugates are in isolated or purified form. As used herein,
"isolated" means separated from other components of (a) a natural
source, such as a plant or animal cell or cell culture, or (b) a
synthetic organic chemical reaction mixture. As used herein,
"purified" means that when isolated, the isolate contains at least
95%, and in another aspect at least 98%, of Exemplary Compound or
Exemplary Conjugate by weight of the isolate.
[0420] Examples of a "hydroxyl protecting group" include, but are
not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether,
tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether,
trimethylsilyl ether, triethylsilyl ether, triisopropyl silyl
ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether,
acetate ester, substituted acetate esters, pivaloate, benzoate,
methanesulfonate and p-toluenesulfonate.
[0421] "Leaving group" refers to a functional group that can be
substituted by another functional group. Such leaving groups are
well known in the art, and examples include, but are not limited
to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl
(mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl
(triflate), and trifluoromethylsulfonate.
[0422] The phrase "pharmaceutically acceptable salt," as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of an Exemplary Compound or Exemplary Conjugate. The
Exemplary Compounds and Exemplary Conjugates contain at least one
amino group, and accordingly acid addition salts can be foamed with
this amino group. Exemplary salts include, but are not limited, to
sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,
nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,
lactate, salicylate, acid citrate, tartrate, oleate, tannate,
pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis -(2-hydroxy-3-naphthoate)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counterion. The counterion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counterion.
[0423] "Pharmaceutically acceptable solvate" or "solvate" refer to
an association of one or more solvent molecules and a compound of
the invention, e.g., an Exemplary Compound or Exemplary Conjugate.
Examples of solvents that form pharmaceutically acceptable solvates
include, but are not limited to, water, isopropanol, ethanol,
methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
[0424] The following abbreviations are used herein and have the
indicated definitions: AE is auristatin E, Boc is
N-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is
1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is
diethylamine, DEAD is diethylazodicarboxylate, DEPC is
diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate,
DIEA is N,N-diisopropylethylamine, dil is dolaisoleuine, DMAP is
4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or
1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is
dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline,
DTNB is 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is
diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ
is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is
electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is
N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high
pressure liquid chromatography, ile is isoleucine, lys is lysine,
MeCN(CH.sub.3CN) is acetonitrile, MeOH is methanol, Mtr is
4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is
(1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is
phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph
is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe is
L-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphonium
hexafluorophosphate, SEC is size-exclusion chromatography, Su is
succinimide, TBTU is O-benzotriazol-1-yl-N,N,N,N-tetramethyluronium
tetrafluoroborate, TFA is trifluoroacetic acid, TLC is thin layer
chromatography, UV is ultraviolet, and val is valine.
[0425] The following linker abbreviations are used herein and have
the indicated definitions: Val Cit is a valine-citrulline,
dipeptide site in protease cleavable linker; PAB is
p-aminobenzylcarbamoyl; (Me)vc is N-methyl-valine citrulline, where
the linker peptide bond has been modified to prevent its cleavage
by cathepsin B; MC(PEG)6-OH is maleimidocaproyl- polyethylene
glycol; SPP is N-Succinimidyl 4-(2-pyridylthio)pentanoate; and SMCC
is N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1
carboxylate.
[0426] The terms "treat" or "treatment," unless otherwise indicated
by context, refer to both therapeutic treatment and prophylactic or
preventative measures, wherein the object is to prevent or slow
down (lessen) an undesired physiological change or disorder, such
as the development or spread of cancer. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. Those in need of treatment include those
already with the condition or disorder as well as those prone to
have the condition or disorder or those in which the condition or
disorder is to be prevented.
[0427] In the context of cancer, the term "treating" includes any
or all of: preventing growth of tumor cells, cancer cells, or of a
tumor; preventing replication of tumor cells or cancer cells,
lessening of overall tumor burden or decreasing the number of
cancerous cells, and ameliorating one or more symptoms associated
with the disease.
[0428] In the context of an autoimmune disease, the term "treating"
includes any or all of: preventing replication of cells associated
with an autoimmune disease state including, but not limited to,
cells that produce an autoimmune antibody, lessening the
autoimmune-antibody burden and ameliorating one or more symptoms of
an autoimmune disease.
[0429] In the context of an infectious disease, the term "treating"
includes any or all of: preventing the growth, multiplication or
replication of the pathogen that causes the infectious disease and
ameliorating one or more symptoms of an infectious disease.
[0430] The following cytotoxic drug abbreviations are used herein
and have the indicated definitions: MMAE is mono-methyl auristatin
E (MW 718); MMAF is
N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine (MW
731.5); MMAF-DMAEA is MMAF with DMAEA (dimethylaminoethylamine) in
an amide linkage to the C-terminal phenylalanine (MW 801.5);
MMAF-TEG is MMAF with tetraethylene glycol esterified to the
phenylalanine; MMAF-NtBu is N-t-butyl, attached as an amide to
C-terminus of MMAF; AEVB is auristatin E valeryl benzylhydrazone,
acid labile linker through the C-terminus of AE (MW 732); and AFP
is Monoamide of p-phenylene diamine with C-terminal Phenylalanine
of Auristatin F (MW 732).
4.2 The Compounds of the Invention
4.2.1 The Compounds of Formula (Ia)
[0431] In one aspect, the invention provides Drug-Linker-Ligand
Conjugates having Formula Ia:
L A.sub.a-W.sub.w-Y.sub.y-D).sub.p Ia
[0432] or a pharmaceutically acceptable salt or solvate thereof
[0433] wherein,
[0434] L- is a Ligand unit;
[0435] -A.sub.a-W.sub.w-Y.sub.y-is a Linker unit (LU), wherein the
Linker unit includes:
[0436] -A- is a Stretcher unit,
[0437] a is 0 or 1,
[0438] each -W- is independently an Amino Acid unit,
[0439] w is an integer ranging from 0 to 12,
[0440] -Y- is a Spacer unit, and
[0441] y is 0, 1 or 2;
[0442] p ranges from 1 to about 20; and
[0443] -D is a Drug unit having the Formulas D.sub.E and
D.sub.F:
##STR00006##
[0444] wherein, independently at each location:
[0445] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0446] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0447] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0448] R.sup.5 is selected from H and methyl;
[0449] or R.sup.4 and R.sup.5 jointly faun a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5
and 6;
[0450] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0451] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0452] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0453] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0454] R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0455] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0456] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0457] m is an integer ranging from 1-1000;
[0458] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0459] R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
[0460] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3-C.sub.1-C.sub.8 alkyl;
[0461] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH;
[0462] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
[0463] n is an integer ranging from 0 to 6.
[0464] In another embodiment, the present invention provides Drug
Compounds having the Formula Ib:
##STR00007##
or pharmaceutically acceptable salts or solvates thereof,
[0465] wherein:
[0466] R.sup.2 is selected from hydrogen and --C.sub.1-C.sub.8
alkyl;
[0467] R.sup.3 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
[0468] R.sup.4 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 jointly, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are
attached;
[0469] R.sup.6 is selected from H and --C.sub.1-C.sub.8 alkyl;
[0470] R.sup.7 is selected from H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
[0471] each R.sup.8 is independently selected from H, --OH,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl);
[0472] R.sup.9 is selected from H and --C.sub.1-C.sub.8 alkyl;
[0473] R.sup.10 is selected from aryl group or --C.sub.3-C.sub.8
heterocycle;
[0474] Z is --O--, --S--, --NH--, or --NR.sup.12--, wherein
R.sup.12 is C.sub.1-C.sub.8 alkyl;
[0475] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
--C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0476] m is an integer ranging from 1-1000;
[0477] R.sup.13 is --C.sub.2-C.sub.8 alkyl;
[0478] R.sup.14 is H or --C.sub.1-C.sub.8 alkyl;
[0479] each occurrence of R.sup.15 is independently H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3-C.sub.1-C.sub.8 alkyl;
[0480] each occurrence of R.sup.16 is independently H,
--C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH; and
[0481] n is an integer ranging from 0 to 6.
[0482] In yet another embodiment, the invention provides
Drug-Linker-Ligand Conjugates having the Formula Ia':
Ab A.sub.a-W.sub.w-Y.sub.y-D).sub.p Formula Ia'
or pharmaceutically acceptable salts or solvates thereof.
[0483] wherein:
[0484] Ab is an antibody,
[0485] A is a Stretcher unit,
[0486] a is 0 or 1,
[0487] each W is independently an Amino Acid unit,
[0488] w is an integer ranging from 0 to 12,
[0489] Y is a Spacer unit, and
[0490] y is 0, 1 or 2,
[0491] p ranges from 1 to about 20, and
[0492] D is a Drug moiety selected from Formulas D.sub.E and
D.sub.F:
##STR00008##
[0493] wherein, independently at each location:
[0494] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0495] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0496] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0497] R.sup.5 is selected from H and methyl;
[0498] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5
and 6;
[0499] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0500] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0501] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0502] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0503] R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0504] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0505] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.n--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0506] m is an integer ranging from 1-1000;
[0507] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0508] R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
[0509] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0510] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH;
[0511] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
[0512] n is an integer ranging from 0 to 6.
[0513] Ab is any antibody covalently attached to one or more drug
units. Ab includes an antibody which binds to CD30, CD40, CD70,
Lewis Y antigen. In another embodiment, Ab does not include an
antibody which binds to an ErbB receptor or to one or more of
receptors
[0514] (1)-(35):
[0515] (1) BMPR1B (bone morphogenetic protein receptor-type IB,
Genbank accession no. NM.sub.--001203);
[0516] (2) El6 (LAT1, SLC7A5, Genbank accession no.
NM.sub.--003486);
[0517] (3) STEAP1 (six transmembrane epithelial antigen of
prostate, Genbank accession no. NM.sub.--012449);
[0518] (4) 0772P (CA125, MUC16, Genbank accession no.
AF361486);
[0519] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
[0520] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family
34 (sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
[0521] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
[0522] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA
2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.
AY358628);
[0523] (9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
[0524] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
[0525] (11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1,
STEAP2, STMP, prostate cancer associated gene 1, prostate cancer
associated protein 1, six transmembrane epithelial antigen of
prostate 2, six transmembrane prostate protein, Genbank accession
no. AF455138);
[0526] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4, Genbank
accession no. NM.sub.--017636);
[0527] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor, Genbank accession no.
NP.sub.--003203 or NM.sub.--003212);
[0528] (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein
Barr virus receptor) or Hs. 73792, Genbank accession no.
M26004);
[0529] (15) CD79b (1 Gb (immunoglobulin-associated beta), B29,
Genbank accession no. NM.sub.--000626);
[0530] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession
no. NM.sub.--030764);
[0531] (17) HER2 (Genbank accession no. M11730);
[0532] (18) NCA (Genbank accession no. M18728);
[0533] (19) MDP (Genbank accession no. BC017023);
[0534] (20) IL20R.alpha. (Genbank accession no. AF184971);
[0535] (21) Brevican (Genbank accession no. AF229053);
[0536] (22) Ephb2R (Genbank accession no. NM.sub.--004442);
[0537] (23) ASLG659 (Genbank accession no. AX092328);
[0538] (24) PSCA (Genbank accession no. AJ297436);
[0539] (25) GEDA (Genbank accession no. AY260763);
[0540] (26) BAFF-R (Genbank accession no. NP 443177.1);
[0541] (27) CD22 (Genbank accession no. NP-001762.1);
[0542] (28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated
alpha, a B cell-specific protein that covalently interacts with Ig
beta (CD79B) and forms a complex on the surface with Ig M
molecules, transduces a signal involved in B-cell differentiation,
Genbank accession No. NP.sub.--001774.1);
[0543] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G
protein-coupled receptor that is activated by the CXCL13 chemokine,
functions in lymphocyte migration and humoral defense, plays a role
in HIV-2 infection and perhaps development of AIDS, lymphoma,
myeloma, and leukemia, Genbank accession No.
NP.sub.--001707.1);
[0544] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia
antigen) that binds peptides and presents them to CD4+ T
lymphocytes, Genbank accession No. NP.sub.--002111.1);
[0545] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel
5, an ion channel gated by extracellular ATP, may be involved in
synaptic transmission and neurogenesis, deficiency may contribute
to the pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
[0546] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2,
Genbank accession No. NP.sub.--001773.1);
[0547] (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane
protein of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
[0548] (34) FCRH1 (Fc receptor-like protein 1, a putative receptor
for the immunoglobulin Fc domain that contains C2 type Ig-like and
ITAM domains, may have a role in B-lymphocyte differentiation,
Genbank accession No. NP.sub.--443170.1); and/or
[0549] (35) IRTA2 (Immunoglobulin superfamily receptor
translocation associated 2, a putative immunoreceptor with possible
roles in B cell development and lymphomagenesis; deregulation of
the gene by translocation occurs in some B cell malignancies,
Genbank accession No. NP.sub.--112571.1).
In one embodiment -Ww- is -Val-Cit-.
[0550] In another embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl. In yet another
embodiment, R.sup.2 and R.sup.6 are each methyl, and R.sup.9 is
--H.
[0551] In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
[0552] In an exemplary embodiment, R.sup.3 and R.sup.4 are each
isopropyl, R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H,
R.sup.7 is sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3,
and R.sup.9 is --H.
[0553] In one embodiment, Z is --O-- or --NH--.
[0554] In one embodiment, R.sup.10 is aryl
[0555] In an exemplary embodiment, R.sup.10 is -phenyl.
[0556] In an exemplary embodiment, when Z is --O--, R.sup.11 is
--H, methyl or t-butyl.
[0557] In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
[0558] In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
-(CH.sub.2).sub.n--SO.sub.3H.
[0559] In one aspect, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, hAC10,
hBR96, hS2C6, h1F6, and h2F2.
[0560] Exemplary embodiments of Formula Ia have the following
structures:
##STR00009##
wherein L is an antibody, Val is valine, and Cit is citrulline.
[0561] The drug loading is represented by p, the average number of
drug molecules per antibody in a molecule (e.g., of Formula Ia, Ia'
and Ic). Drug loading may range from 1 to 20 drugs (D) per Ligand
(eg. Ab or mAb). Compositions of Formula Ia and Formula Ia' include
collections of antibodies conjugated with a range of drugs, from 1
to 20. The average number of drugs per antibody in preparation of
conjugation reactions may be characterized by conventional means
such as mass spectroscopy, ELISA assay, and HPLC. The quantitative
distribution of Ligand-Drug-Conjugates in terms of p may also be
determined. In some instances, separation, purification, and
characterization of homogeneous Ligand-Drug-conjugates where p is a
certain value from Ligand-Drug-Conjugates with other drug loadings
may be achieved by means such as reverse phase HPLC or
electrophoresis.
4.2.2 The Drug Compounds of Formula (Ib)
[0562] In another aspect, the present invention provides Drug
Compounds having the Formula (Ib):
##STR00010##
[0563] or a pharmaceutically acceptable salt or solvate
thereof,
[0564] wherein:
[0565] R.sup.2 is selected from -hydrogen and --C.sub.1-C.sub.8
alkyl;
[0566] R.sup.3 is selected from -hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
[0567] R.sup.4 is selected from -hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 jointly, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are
attached;
[0568] R.sup.6 is selected from --H and --C.sub.1-C.sub.8
alkyl;
[0569] R.sup.7 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
[0570] each R.sup.8 is independently selected from --H, --OH,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl);
[0571] R.sup.9 is selected from --H and --C.sub.1-C.sub.8
alkyl;
[0572] R.sup.10 is selected from aryl group or --C.sub.3-C.sub.8
heterocycle;
[0573] Z is --O--, --S--, --NH--, or --NR.sup.12--, wherein
R.sup.12 is C.sub.1-C.sub.8 alkyl;
[0574] R.sup.11 is selected from --H, C.sub.1-C.sub.20 alkyl, aryl,
--C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0575] m is an integer ranging from 1-1000;
[0576] R.sup.13 is --C.sub.2-C.sub.8 alkyl;
[0577] R.sup.14 is --H or --C.sub.1-C.sub.8 alkyl;
[0578] each occurrence of R.sup.15 is independently --H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0579] each occurrence of R.sup.16 is independently --H,
--C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH; and
[0580] n is an integer ranging from 0 to 6.
[0581] In one embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl.
[0582] In another embodiment, R.sup.2 and R.sup.6 are each methyl,
and R.sup.9 is --H.
[0583] In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
[0584] In an exemplary embodiment, R.sup.3 and R.sup.4 are each
isopropyl, R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H,
R.sup.7 is sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3,
and R.sup.9 is --H.
[0585] In one embodiment, Z is --O-- or --NH--.
[0586] In one embodiment, R.sup.10 is aryl
[0587] In an exemplary embodiment, R.sup.10 is -phenyl.
[0588] In an exemplary embodiment, when Z is --O--, R.sup.11 is
--H, methyl or t-butyl.
[0589] In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
[0590] In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
[0591] Illustrative Compounds of Formula (Ib), each of which may be
used as drug moieties (D) in ADC, include compounds having the
following structures:
##STR00011## ##STR00012##
and pharmaceutically acceptable salts or solvates thereof.
The Compounds of Formula (Ic)
[0592] In another aspect, the invention provides antibody-drug
conjugate compounds (ADC) having Formula Ic:
Ab A.sub.a-W.sub.w-Y.sub.yD).sub.p Ic
comprising an antibody covalently attached to one or more drug
units (moieties). The antibody-drug conjugate compounds include
pharmaceutically acceptable salts or solvates thereof.
[0593] Formula Ic compounds are defined wherein:
[0594] Ab is an antibody which binds to one or more
tumor-associated antigen receptors (1)-(35):
[0595] (1) BMPR1B (bone morphogenetic protein receptor-type IB,
Genbank accession no. NM.sub.--001203);
[0596] (2) E16 (LAT1, SLC7A5, Genbank accession no.
NM.sub.--003486);
[0597] (3) STEAP1 (six transmembrane epithelial antigen of
prostate, Genbank accession no. NM.sub.--012449);
[0598] (4) 0772P (CA125, MUC16, Genbank accession no.
AF361486);
[0599] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
[0600] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family
34 (sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
[0601] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
[0602] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA
2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.
AY358628);
[0603] (9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
[0604] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
[0605] (11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1,
STEAP2, STMP, prostate cancer associated gene 1, prostate cancer
associated protein 1, six transmembrane epithelial antigen of
prostate 2, six transmembrane prostate protein, Genbank accession
no. AF455138);
[0606] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4, Genbank
accession no. NM.sub.--017636);
[0607] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor, Genbank accession no.
NP.sub.--003203 or NM.sub.--003212);
[0608] (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein
Barr virus receptor) or Hs. 73792 Genbank accession no.
M26004);
[0609] (15) CD79b (CD79B, CD79.beta., IGb
(immunoglobulin-associated beta), B29, Genbank accession no.
NM.sub.--000626);
[0610] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession
no. NM.sub.--030764);
[0611] (17) HER2 (Genbank accession no. M11730);
[0612] (18) NCA (Genbank accession no. M18728);
[0613] (19) MDP (Genbank accession no. BC017023);
[0614] (20) IL20R.alpha. (Genbank accession no. AF184971);
[0615] (21) Brevican (Genbank accession no. AF229053);
[0616] (22) Ephb2R (Genbank accession no. NM.sub.--004442);
[0617] (23) ASLG659 (Genbank accession no. AX092328);
[0618] (24) PSCA (Genbank accession no. AJ297436);
[0619] (25) GEDA (Genbank accession no. AY260763;
[0620] (26) BAFF--R (B cell-activating factor receptor, BLyS
receptor 3, BR3, NP.sub.--443177.1);
[0621] (27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1);
[0622] (28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation, Genbank
accession No. NP.sub.--001774.1);
[0623] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G
protein-coupled receptor that is activated by the CXCL13 chemokine,
functions in lymphocyte migration and humoral defense, plays a role
in HIV-2 infection and perhaps development of AIDS, lymphoma,
myeloma, and leukemia, Genbank accession No.
NP.sub.--001707.1);
[0624] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia
antigen) that binds peptides and presents them to CD4+ T
lymphocytes, Genbank accession No. NP.sub.--002111.1);
[0625] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel
5, an ion channel gated by extracellular ATP, may be involved in
synaptic transmission and neurogenesis, deficiency may contribute
to the pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
[0626] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2,
Genbank accession No. NP.sub.--001773.1);
[0627] (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane
protein of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
[0628] (34) FCRH1 (Fc receptor-like protein 1, a putative receptor
for the immunoglobulin Fc domain that contains C2 type Ig-like and
ITAM domains, may have a role in B-lymphocyte differentiation,
Genbank accession No. NP.sub.--443170.1); and
[0629] (35) IRTA2 (Immunoglobulin superfamily receptor
translocation associated 2, a putative immunoreceptor with possible
roles in B cell development and lymphomagenesis; deregulation of
the gene by translocation occurs in some B cell malignancies,
Genbank accession No. NP.sub.--112571.1).
[0630] A is a Stretcher unit,
[0631] a is 0 or 1,
[0632] each W is independently an Amino Acid unit,
[0633] w is an integer ranging from 0 to 12,
[0634] Y is a Spacer unit, and
[0635] y is 0, 1 or 2,
[0636] p ranges from 1 to about 8, and
[0637] D is a Drug moiety selected from Formulas D.sub.E and
D.sub.F:
##STR00013##
wherein the wavy line of D.sub.E and D.sub.E indicates the covalent
attachment site to A, W, or Y, and independently at each
location:
[0638] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0639] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0640] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0641] R.sup.5 is selected from H and methyl;
[0642] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5
and 6;
[0643] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0644] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0645] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0646] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0647] R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0648] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0649] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0650] m is an integer ranging from 1-1000;
[0651] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0652] R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
[0653] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0654] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH;
[0655] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
[0656] n is an integer ranging from 0 to 6.
[0657] In one embodiment -Ww- is -Val-Cit-.
[0658] In another embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl.
[0659] In yet another embodiment, R.sup.2 and R.sup.6 are each
methyl, and R.sup.9 is --H.
[0660] In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
[0661] In an exemplary embodiment, R.sup.3 and R.sup.4 are each
isopropyl, R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H,
R.sup.7 is sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3,
and R.sup.9 is --H.
[0662] In one embodiment, Z is --O-- or --NH--.
[0663] In one embodiment, R.sup.10 is aryl.
[0664] In an exemplary embodiment, R.sup.10 is -phenyl.
[0665] In an exemplary embodiment, when Z is --O--, R.sup.11 is
--H, methyl or t-butyl.
[0666] In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.m--COOH.
[0667] In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.nSO.sub.3H.
[0668] Exemplary embodiments of Formula Ic ADC have the following
structures:
##STR00014##
wherein Ab is an antibody which binds to one or more
tumor-associated antigen receptors (1)-(35); Val is valine; and Cit
is citrulline.
[0669] The drug loading is represented by p, the average number of
drugs per antibody in a molecule of Formula I. Drug loading may
range from 1 to 20 drugs (D) per antibody (Ab or mAb). Compositions
of ADC of Formula I include collections of antibodies conjugated
with a range of drugs, from 1 to 20. The average number of drugs
per antibody in preparations of ADC from conjugation reactions may
be characterized by conventional means such as UV/visible
spectroscopy, mass spectrometry, ELISA assay, and HPLC. The
quantitative distribution of ADC in terms of p may also be
determined. In some instances, separation, purification, and
characterization of homogeneous ADC where p is a certain value from
ADC with other drug loadings may be achieved by means such as
reverse phase HPLC or electrophoresis.
[0670] For some antibody drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached.
[0671] Typically, fewer than the theoretical maximum of drug
moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, many lysine
residues that do not react with the drug-linker intermediate or
linker reagent. Only the most reactive lysine groups may react with
an amine-reactive linker reagent. Generally, antibodies do not
contain many, if any, free and reactive cysteine thiol groups which
may be linked to a drug moiety. Most cysteine thiol residues in the
antibodies of the compounds of the invention exist as disulfide
bridges and must be reduced with a reducing agent such as
dithiothreitol (DTT). Additionally, the antibody must be subjected
to denaturing conditions to reveal reactive nucleophilic groups
such as lysine or cysteine. The loading (drug/antibody ratio) of an
ADC may be controlled in several different manners, including: (i)
limiting the molar excess of drug-linker intermediate or linker
reagent relative to antibody, (ii) limiting the conjugation
reaction time or temperature, and (iii) partial or limiting
reductive conditions for cysteine thiol modification.
[0672] It is to be understood that where more than one nucleophilic
group reacts with a drug-linker intermediate, or linker reagent
followed by drug moiety reagent, then the resulting product is a
mixture of ADC compounds with a distribution of one or more drug
moieties attached to an antibody. The average number of drugs per
antibody may be calculated from the mixture by dual ELISA antibody
assay, specific for antibody and specific for the drug. Individual
ADC molecules may be identified in the mixture by mass
spectroscopy, and separated by HPLC, e.g., hydrophobic interaction
chromatography ("Effect of drug loading on the pharmacology,
pharmacokinetics, and toxicity of an anti-CD30-antibody-drug
conjugate", Hamblett, K. J., et al, Abstract No. 624, American
Association for Cancer Research; 2004Annual Meeting, Mar. 27-31,
2004, Proceedings of the AACR, Volume 45, March 2004; "Controlling
the Location of Drug Attachment in Antibody-Drug Conjugates",
Alley, S. C., et al, Abstract No. 627, American Association for
Cancer Research; 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings
of the AACR, Volume 45, March 2004). Thus, a homogeneous ADC with a
single loading value may be isolated from the conjugation mixture
by electrophoresis or chromatography.
4.3 The Linker Unit
[0673] A "Linker unit" (LU) is a bifunctional compound which can be
used to link a Drug unit and an Ligand unit to form
Drug-Linker-Ligand Conjugates, or which are useful in the formation
of immunoconjugates directed against tumor associated antigens.
Such immunoconjugates allow the selective delivery of toxic drugs
to tumor cells. In one embodiment, the Linker unit of the
Drug-Linker Compound and Drug-Linker-Ligand Conjugate has the
formula:
-A.sub.a-W.sub.w-Y.sub.y-
wherein:
[0674] -A- is a Stretcher unit;
[0675] a is 0 or 1;
[0676] each -W- is independently an Amino Acid unit;
[0677] w is independently an integer ranging from 0 to 12;
[0678] -Y- is a Spacer unit; and
[0679] y is 0, 1 or 2.
[0680] In the Drug-Linker-Ligand Conjugate, the Linker is capable
of linking the Drug moiety and the Ligand unit.
4.3.1 The Stretcher Unit
[0681] The Stretcher unit (-A-), when present, is capable of
linking a Ligand unit to an amino acid unit (-W-). In this regard a
Ligand (L) has a functional group that can form a bond with a
functional group of a Stretcher. Useful functional groups that can
be present on a ligand, either naturally or via chemical
manipulation include, but are not limited to, sulfhydryl (--SH),
amino, hydroxyl, carboxy, the anomeric hydroxyl group of a
carbohydrate, and carboxyl. In one aspect, the Ligand functional
groups are sulfhydryl and amino. Sulfhydryl groups can be generated
by reduction of an intramolecular disulfide bond of a Ligand.
Alternatively, sulfhydryl groups can be generated by reaction of an
amino group of a lysine moiety of a Ligand using 2-iminothiolane
(Traut's reagent) or another sulfhydryl generating reagent.
[0682] In one embodiment, the Stretcher unit forms a bond with a
sulfur atom of the Ligand unit. The sulfur atom can be derived from
a sulfhydryl group of a Ligand. Representative Stretcher units of
this embodiment are depicted within the square brackets of Formulas
IIIa and IIIb, wherein L-, -W-, -Y-, -D, w and y are as defined
above, and R.sup.17 is selected from --C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-,
-arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 carbocyclo)-, --(C.sub.3-C.sub.8
carbocyclo)-C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
heterocyclo-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10. It is to be understood from all the exemplary
embodiments of Formula Ia, such as III-VI, that even where not
denoted expressly, from 1 to 20 drug moieties are linked to a
Ligand (p=1-20).
##STR00015##
[0683] An illustrative Stretcher unit is that of Formula Ma wherein
R.sup.17 is --(CH.sub.2).sub.5--:
##STR00016##
[0684] Another illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r
is 2:
##STR00017##
[0685] Still another illustrative Stretcher unit is that of Formula
IIIb wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00018##
[0686] In another embodiment, the Stretcher unit is linked to the
Ligand unit via a disulfide bond between a sulfur atom of the
Ligand unit and a sulfur atom of the Stretcher unit. A
representative Stretcher unit of this embodiment is depicted within
the square brackets of Formula IV, wherein R.sup.17, L-, -W-, -Y-,
-D, w and y are as defined above.
##STR00019##
[0687] In yet another embodiment, the reactive group of the
Stretcher contains a reactive site that can form a bond with a
primary or secondary amino group of a Ligand. Example of these
reactive sites include, but are not limited to, activated esters
such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl
esters, tetrafluorophenyl esters, anhydrides, acid chlorides,
sulfonyl chlorides, isocyanates and isothiocyanates. Representative
Stretcher units of this embodiment are depicted within the square
brackets of Formulas Va and Vb, wherein --R.sup.17--, L-, -W-, -Y-,
-D, w and y are as defined above;
##STR00020##
[0688] In yet another aspect, the reactive group of the Stretcher
contains a reactive site that is reactive to a modified
carbohydrate's (--CHO) group that can be present on a Ligand. For
example, a carbohydrate can be mildly oxidized using a reagent such
as sodium periodate and the resulting (--CHO) unit of the oxidized
carbohydrate can be condensed with a Stretcher that contains a
functionality such as a hydrazide, an oxime, a primary or secondary
amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate,
and an arylhydrazide such as those described by Kaneko, T. et al.
(1991) Bioconjugate Chem 2:133-41. Representative Stretcher units
of this embodiment are depicted within the square brackets of
Formulas VIa, VIb, and VIc, wherein --R.sup.17--, L-, -W-, -Y-, -D,
w and y are as defined above.
##STR00021##
4.3.2 The Amino Acid Unit
[0689] The Amino Acid unit (-W-), when present, links the Stretcher
unit to the Spacer unit if the Spacer unit is present, links the
Stretcher unit to the Drug moiety if the Spacer unit is absent, and
links the Ligand unit to the Drug unit if the Stretcher unit and
Spacer unit are absent.
[0690] W.sub.w- is a dipeptide, tripeptide, tetrapeptide,
pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,
decapeptide, undecapeptide or dodecapeptide unit. Each -W- unit
independently has the formula denoted below in the square brackets,
and w is an integer ranging from 0 to 12:
##STR00022##
wherein R.sup.19 is hydrogen, methyl, isopropyl, isobutyl,
sec-butyl, benzyl, p-hydroxybenzyl, --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2CONH.sub.2, --CH.sub.2COOH,
--CH.sub.2CH.sub.2CONH.sub.2, --CH.sub.2CH.sub.2COOH,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.3NHCOCH.sub.3, --(CH.sub.2).sub.3NHCHO,
--(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.4NH.sub.2,
--(CH.sub.2).sub.4NHCOCH.sub.3, --(CH.sub.2).sub.4NHCHO,
--(CH.sub.2).sub.3NHCONH.sub.2, --(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR00023##
[0691] The Amino Acid unit can be enzymatically cleaved by one or
more enzymes, including a tumor-associated protease, to liberate
the Drug unit (-D), which in one embodiment is protonated in vivo
upon release to provide a Drug (D). Illustrative W.sub.w units are
represented by formulas (VII)-(IX):
##STR00024##
wherein R.sup.20 and R.sup.21 are as follows:
TABLE-US-00001 R.sup.20 R.sup.21 benzyl (CH.sub.2).sub.4NH.sub.2;
methyl (CH.sub.2).sub.4NH.sub.2; isopropyl
(CH.sub.2).sub.4NH.sub.2; isopropyl (CH.sub.2).sub.3NHCONH.sub.2;
benzyl (CH.sub.2).sub.3NHCONH.sub.2; isobutyl
(CH.sub.2).sub.3NHCONH.sub.2; sec-butyl
(CH.sub.2).sub.3NHCONH.sub.2; ##STR00025##
(CH.sub.2).sub.3NHCONH.sub.2; benzyl methyl; and benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2; (VIII) ##STR00026##
wherein R.sup.20, R.sup.21 and R.sup.22 are as follows:
TABLE-US-00002 R.sup.20 R.sup.21 R.sup.22 benzyl benzyl
(CH.sub.2).sub.4NH.sub.2; isopropyl benzyl
(CH.sub.2).sub.4NH.sub.2; and H benzyl (CH.sub.2).sub.4NH.sub.2;
(IX) ##STR00027##
wherein R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are as
follows:
TABLE-US-00003 R.sup.20 R.sup.21 R.sup.22 R.sup.23 H benzyl
isobutyl H; and methyl isobutyl methyl isobutyl.
[0692] Exemplary Amino Acid units include, but are not limited to,
units of formula (VII) where: R.sup.20 is benzyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.21 isopropyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.20 isopropyl and R.sup.21 is
--(CH.sub.2).sub.3NHCONH.sub.2. Another exemplary Amino Acid unit
is a unit of formula (VIII) wherein R.sup.20 is benzyl, R.sup.21 is
benzyl, and R.sup.22 is --(CH.sub.2).sub.4NH.sub.2.
[0693] Useful -W.sub.w-units can be designed and optimized in their
selectivity for enzymatic cleavage by a particular enzymes, for
example, a tumor-associated protease. In one embodiment, a
-W.sub.w-unit is that whose cleavage is catalyzed by cathepsin B, C
and D, or a plasmin protease.
[0694] In one embodiment, -W.sub.w- is a dipeptide, tripeptide,
tetrapeptide or pentapeptide.
[0695] When R.sup.19, R.sup.20, R.sup.21, R.sup.22 or R.sup.23 is
other than hydrogen, the carbon atom to which R.sup.19, R.sup.20,
R.sup.21, R.sup.22 or R.sup.23 is attached is chiral.
[0696] Each carbon atom to which R.sup.19, R.sup.20, R.sup.21,
R.sup.22 or R.sup.23 is attached is independently in the (S) or (R)
configuration.
[0697] In one aspect of the Amino Acid unit, the Amino Acid unit is
valine-citrulline. In another aspect, the Amino Acid unit is
phenylalanine-lysine (i.e. fk). In yet another aspect of the Amino
Acid unit, the Amino Acid unit is N-methylvaline-citrulline. In yet
another aspect, the Amino Acid unit is 5-aminovaleric acid, homo
phenylalanine lysine, tetraisoquinolinecarboxylate lysine,
cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine
lysine, glycine serine valine glutamine and isonepecotic acid.
[0698] In certain embodiments, the Amino Acid unit can comprise
natural amino acids. In other embodiments, the Amino Acid unit can
comprise non-natural amino acids.
4.3.3 The Spacer Unit
[0699] The Spacer unit (-Y-), when present, links an Amino Acid
unit to the Drug moiety when an Amino Acid unit is present.
Alternately, the Spacer unit links the Stretcher unit to the Drug
moiety when the Amino Acid unit is absent. The Spacer unit also
links the Drug moiety to the Ligand unit when both the Amino Acid
unit and Stretcher unit are absent.
[0700] Spacer units are of two general types: self-immolative and
non self-immolative. A non self-immolative Spacer unit is one in
which part or all of the Spacer unit remains bound to the Drug
moiety after cleavage, particularly enzymatic, of an Amino Acid
unit from the Drug-Linker-Ligand Conjugate or the Drug-Linker
Compound. Examples of a non self-immolative Spacer unit include,
but are not limited to a (glycine-glycine) Spacer unit and a
glycine Spacer unit (both depicted in FIG. 20) (infra). When an
Exemplary Compound containing a glycine-glycine Spacer unit or a
glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell
associated-protease, a cancer-cell-associated protease or a
lymphocyte-associated protease, a glycine-glycine-Drug moiety or a
glycine-Drug moiety is cleaved from L-A.sub.a-W.sub.w-. In one
embodiment, an independent hydrolysis reaction takes place within
the target cell, cleaving the glycine--Drug moiety bond and
liberating the Drug.
[0701] In another embodiment, -Y.sub.y- is a p-aminobenzyl alcohol
(PAB) unit (see FIGS. 21 and 22) whose phenylene portion is
substituted with Q.sub.m wherein Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
##STR00028##
[0702] In one embodiment, a non self-immolative Spacer unit (-Y-)
is -Gly-Gly-. In another embodiment, a non self-immolative the
Spacer unit (-Y-) is -Gly-.
[0703] In one embodiment, a Drug-Linker Compound or a Drug-Linker
Ligand Conjugate is provided in which the Spacer unit is absent
(y=0), or a pharmaceutically acceptable salt or solvate
thereof.
[0704] Alternatively, an Exemplary Compound containing a
self-immolative Spacer unit can release -D without the need for a
separate hydrolysis step. In this embodiment, -Y-is a PAB group
that is linked to -W.sub.w-via the amino nitrogen atom of the PAB
group, and connected directly to -D via a carbonate, carbamate or
ether group. Without being bound by any particular theory or
mechanism, FIG. 21 depicts a possible mechanism of Drug release of
a PAB group which is attached directly to -D via a carbamate or
carbonate group espoused by Toki et al. (2002) J. Org. Chem.
67:1866-1872.
##STR00029##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p
ranges from 1 to about 20.
[0705] Without being bound by any particular theory or mechanism,
FIG. 22 depicts a possible mechanism of Drug release of a PAB group
which is attached directly to -D via an ether or amine linkage.
##STR00030##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p
ranges from 1 to about 20.
[0706] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically similar
to the PAB group such as 2-aminoimidazol-5-methanol derivatives
(Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho or
para-aminobenzylacetals. Spacers can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al.,
Chemistry Biology, 1995, 2, 223), appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J.
Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid
amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867).
Elimination of amine-containing drugs that are substituted at the
a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27,
1447) are also examples of self-immolative spacer useful in
Exemplary Compounds.
[0707] In one embodiment, the Spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit as depicted in FIG. 23, which
can be used to incorporate and release multiple drugs.
##STR00031##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is
0 or 1; and p ranges raging from 1 to about 20.
[0708] In one embodiment, the -D moieties are the same. In yet
another embodiment, the -D moieties are different.
[0709] In one aspect, Spacer units (-Y.sub.y-) are represented by
Formulas (X) --(XII):
##STR00032##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; and m is an integer ranging from
0-4;
##STR00033##
[0710] Embodiments of the Formula Ia' and Ic antibody-drug
conjugate compounds include:
##STR00034##
wherein w and y are each 0,
##STR00035##
4.4 The Drug Unit
Moiety
[0711] The drug moiety (D) of the antibody drug conjugates (ADC)
are of the dolastatin/auristatin type (U.S. Pat. Nos. 5,635,483;
5,780,588) which have been shown to interfere with microtubule
dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke
et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)
and have anticancer (U.S. Pat. No. 5,663,149) and antifungal
activity (Pettit et al. (1998) Antimicrob. Agents Chemother.
42:2961-2965)
[0712] D is a Drug unit (moiety) having a nitrogen atom that can
form a bond with the Spacer unit when y=1 or 2, with the C-terminal
carboxyl group of an Amino Acid unit when y=0, with the carboxyl
group of a Stretcher unit when w and y=0, and with the carboxyl
group of a Drug unit when a, w, and y=0. It is to be understood
that the terms "drug unit" and "drug moiety" are synonymous and
used interchangeably herein.
[0713] In one embodiment, -D is either formula D.sub.E or
D.sub.F:
##STR00036##
wherein, independently at each location:
[0714] R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
[0715] R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0716] R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0717] R.sup.5 is selected from H and methyl;
[0718] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl
and C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5
and 6;
[0719] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
[0720] R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle);
[0721] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle and
O--(C.sub.1-C.sub.8 alkyl);
[0722] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
[0723] R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle;
[0724] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
[0725] R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0726] m is an integer ranging from 1-1000;
[0727] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0728] R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
[0729] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
[0730] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.8 alkyl, or --(CH.sub.2).sub.n--COOH;
[0731] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
[0732] n is an integer ranging from 0 to 6.
[0733] In one embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is H, and R.sup.7 is sec-butyl.
[0734] In another embodiment, R.sup.2 and R.sup.6 are each methyl,
and R.sup.9 is H.
[0735] In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
[0736] In an exemplary embodiment, R.sup.3 and R.sup.4 are each
isopropyl, R.sup.2 and R.sup.6 are each methyl, R.sup.5 is H,
R.sup.7 is sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3,
and R.sup.9 is H.
[0737] In one embodiment, Z is --O-- or --NH--.
[0738] In one embodiment, R.sup.10 is aryl
[0739] In an exemplary embodiment, R.sup.10 is -phenyl.
[0740] In an exemplary embodiment, when Z is --O--, R.sup.11 is H,
methyl or t-butyl.
[0741] In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
[0742] In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
[0743] Illustrative Drug units (-D) include the drug units having
the following structures:
##STR00037## ##STR00038##
and pharmaceutically acceptable salts or solvates thereof.
[0744] In one aspect, hydrophilic groups, such as but not limited
to triethylene glycol esters (TEG), as shown above, can be attached
to the Drug Unit at R.sup.11. Without being bound by theory, the
hydrophilic groups assist in the internalization and
non-agglomeration of the Drug Unit.
4.5 The Ligand Unit
[0745] The Ligand unit (L-) includes within its scope any unit of a
Ligand (L) that binds or reactively associates or complexes with a
receptor, antigen or other receptive moiety associated with a given
target-cell population. A Ligand is a molecule that binds to,
complexes with, or reacts with a moiety of a cell population sought
to be therapeutically or otherwise biologically modified. In one
aspect, the Ligand unit acts to deliver the Drug unit to the
particular target cell population with which the Ligand unit
reacts. Such Ligands include, but are not limited to, large
molecular weight proteins such as, for example, full-length
antibodies, antibody fragments, smaller molecular weight proteins,
polypeptide or peptides, lectins, glycoproteins, non-peptides,
vitamins, nutrient-transport molecules (such as, but not limited
to, transferrin), or any other cell binding molecule or
substance.
[0746] A Ligand unit can form a bond to a Stretcher unit, an Amino
Acid unit, a Spacer Unit, or a Drug Unit. A Ligand unit can form a
bond to a Linker unit via a heteroatom of the Ligand. Heteroatoms
that may be present on a Ligand unit include sulfur (in one
embodiment, from a sulfhydryl group of a Ligand), oxygen (in one
embodiment, from a carbonyl, carboxyl or hydroxyl group of a
Ligand) and nitrogen (in one embodiment, from a primary or
secondary amino group of a Ligand). These heteroatoms can be
present on the Ligand in the Ligand's natural state, for example a
naturally-occurring antibody, or can be introduced into the Ligand
via chemical modification.
[0747] In one embodiment, a Ligand has a sulfhydryl group and the
Ligand bonds to the Linker unit via the sulfhydryl group's sulfur
atom.
[0748] In yet another aspect, the Ligand has one or more lysine
residues that can be chemically modified to introduce one or more
sulfhydryl groups. The Ligand unit bonds to the Linker unit via the
sulfhydryl group's sulfur atom. The reagents that can be used to
modify lysines include, but are not limited to, N-succinimidyl
S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride
(Traut's Reagent).
[0749] In another embodiment, the Ligand can have one or more
carbohydrate groups that can be chemically modified to have one or
more sulfhydryl groups. The Ligand unit bonds to the Linker Unit,
such as the Stretcher Unit, via the sulfhydryl group's sulfur
atom.
[0750] In yet another embodiment, the Ligand can have one or more
carbohydrate groups that can be oxidized to provide an aldehyde
(--CHO) group (see, for e.g., Laguzza, et al., J. Med. Chem. 1989,
32(3), 548-55). The corresponding aldehyde can form a bond with a
Reactive Site on a Stretcher. Reactive sites on a Stretcher that
can react with a carbonyl group on a Ligand include, but are not
limited to, hydrazine and hydroxylamine. Other protocols for the
modification of proteins for the attachment or association of Drug
Units are described in Coligan et al., Current Protocols in Protein
Science, vol. 2, John Wiley & Sons (2002), incorporated herein
by reference.
[0751] Useful non-immunoreactive protein, polypeptide, or peptide
Ligands include, but are not limited to, transferrin, epidermal
growth factors ("EGF"), bombesin, gastrin, gastrin-releasing
peptide, platelet-derived growth factor, IL-2, IL-6, transforming
growth factors ("TGF"), such as TGF-.alpha. and TGF-.beta.,
vaccinia growth factor ("VGF"), insulin and insulin-like growth
factors I and II, lectins and apoprotein from low density
lipoprotein.
[0752] Useful polyclonal antibodies are heterogeneous populations
of antibody molecules derived from the sera of immunized animals.
Various procedures well known in the art may be used for the
production of polyclonal antibodies to an antigen-of-interest. For
example, for the production of polyclonal antibodies, various host
animals can be immunized by injection with an antigen of interest
or derivative thereof, including but not limited to rabbits, mice,
rats, and guinea pigs. Various adjuvants may be used to increase
the immunological response, depending on the host species, and
including but not limited to Freund's (complete and incomplete)
adjuvant, mineral gels such as aluminum hydroxide, surface active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol,
and potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art.
[0753] Useful monoclonal antibodies are homogeneous populations of
antibodies to a particular antigenic determinant (e.g., a cancer
cell antigen, a viral antigen, a microbial antigen, a protein, a
peptide, a carbohydrate, a chemical, nucleic acid, or fragments
thereof). A monoclonal antibody (mAb) to an antigen-of-interest can
be prepared by using any technique known in the art which provides
for the production of antibody molecules by continuous cell lines
in culture. These include, but are not limited to, the hybridoma
technique originally described by Kohler and Milstein (1975, Nature
256, 495-497), the human B cell hybridoma technique (Kozbor et al.,
1983, Immunology Today 4: 72), and the EBV-hybridoma technique
(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, Inc., pp. 77-96). Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any
subclass thereof. The hybridoma producing the mAbs of use in this
invention may be cultivated in vitro or in vivo.
[0754] Useful monoclonal antibodies include, but are not limited
to, human monoclonal antibodies, humanized monoclonal antibodies,
antibody fragments, or chimeric human-mouse (or other species)
monoclonal antibodies. Human monoclonal antibodies may be made by
any of numerous techniques known in the art (e.g., Teng et al.,
1983, Proc. Natl. Acad. Sci. USA. 80, 7308-7312; Kozbor et al.,
1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth.
Enzymol. 92, 3-16).
[0755] The antibody can also be a bispecific antibody. Methods for
making bispecific antibodies are known in the art. Traditional
production of full-length bispecific antibodies is based on the
coexpression of two immunoglobulin heavy chain-light chain pairs,
where the two chains have different specificities (Milstein et al.,
1983, Nature 305:537-539). Because of the random assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of
which only one has the correct bispecific structure. Similar
procedures are disclosed in International Publication No. WO
93/08829, and in Traunecker et al., EMBO J. 10:3655-3659
(1991).
[0756] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. It is preferred to have the first heavy-chain
constant region (C.sub.H1) containing the site necessary for light
chain binding, present in at least one of the fusions. Nucleic
acids with sequences encoding the immunoglobulin heavy chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. This provides for great flexibility
in adjusting the mutual proportions of the three polypeptide
fragments in embodiments when unequal ratios of the three
polypeptide chains used in the construction provide the optimum
yields. It is, however, possible to insert the coding sequences for
two or all three polypeptide chains in one expression vector when
the expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0757] In an embodiment of this approach, the bispecific antibodies
have a hybrid immunoglobulin heavy chain with a first binding
specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. This asymmetric structure facilitates the separation
of the desired bispecific compound from unwanted immunoglobulin
chain combinations, as the presence of an immunoglobulin light
chain in only one half of the bispecific molecule provides for a
facile way of separation (International Publication No. WO
94/04690) which is incorporated herein by reference in its
entirety.
[0758] For further details for generating bispecific antibodies
see, for example, Suresh et al., Methods in Enzymology, 1986,
121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961;
Carter et al., 1992, Bio/Technology 10:163-167; Carter et al.,
1995, J. of Hematotherapy 4:463-470; Merchant et al., 1998, Nature
Biotechnology 16:677-681. Using such techniques, bispecific
antibodies can be prepared for use in the treatment or prevention
of disease as defined herein.
[0759] Bifunctional antibodies are also described, in European
Patent Publication No. EPA 0 105 360. As disclosed in this
reference, hybrid or bifunctional antibodies can be derived either
biologically, i.e., by cell fusion techniques, or chemically,
especially with cross-linking agents or disulfide-bridge forming
reagents, and may comprise whole antibodies or fragments thereof.
Methods for obtaining such hybrid antibodies are disclosed for
example, in International Publication WO 83/03679, and European
Patent Publication No. EPA 0 217 577, both of which are
incorporated herein by reference.
[0760] The antibody can be a functionally active fragment,
derivative or analog of an antibody that immunospecifically binds
to cancer cell antigens, viral antigens, or microbial antigens or
other antibodies bound to tumor cells or matrix. In this regard,
"functionally active" means that the fragment, derivative or analog
is able to elicit anti-anti-idiotype antibodies that recognize the
same antigen that the antibody from which the fragment, derivative
or analog is derived recognized. Specifically, in an exemplary
embodiment the antigenicity of the idiotype of the immunoglobulin
molecule can be enhanced by deletion of framework and CDR sequences
that are C-terminal to the CDR sequence that specifically
recognizes the antigen. To determine which CDR sequences bind the
antigen, synthetic peptides containing the CDR sequences can be
used in binding assays with the antigen by any binding assay method
known in the art (e.g., the BIA core assay) (See, for e.g., Kabat
et al., 1991, Sequences of Proteins of Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E
et al., 1980, J. of Immunology 125(3):961-969).
[0761] Other useful antibodies include fragments of antibodies such
as, but not limited to, F(ab').sub.2 fragments, which contain the
variable region, the light chain constant region and the CH1 domain
of the heavy chain can be produced by pepsin digestion of the
antibody molecule, and Fab fragments, which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragments. Other
useful antibodies are heavy chain and light chain dimers of
antibodies, or any minimal fragment thereof such as Fvs or single
chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.
4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-54), or any other molecule with the same specificity
as the antibody.
[0762] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are useful antibodies. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal and human immunoglobulin constant regions.
(See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et
al., U.S. Pat. No. 4,816,397, which are incorporated herein by
reference in their entirety.) Humanized antibodies are antibody
molecules from non-human species having one or more complementarity
determining regions (CDRs) from the non-human species and a
framework region from a human immunoglobulin molecule. (See, e.g.,
Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by
reference in its entirety.) Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in International
Publication No. WO 87/02671; European Patent Publication No.
184,187; European Patent Publication No. 171496; European Patent
Publication No. 173494; International Publication No. WO 86/01533;
U.S. Pat. No. 4,816,567; European Patent Publication No. 12,023;
Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc.
Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.
139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA
84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood
et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl.
Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207;
Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539;
Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al., 1988, J. Immunol.
141:4053-4060; each of which is incorporated herein by reference in
its entirety.
[0763] Completely human antibodies are particularly desirable and
can be produced using transgenic mice that are incapable of
expressing endogenous immunoglobulin heavy and light chains genes,
but which can express human heavy and light chain genes. The
transgenic mice are immunized in the normal fashion with a selected
antigen, e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
using conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which is
incorporated herein by reference in its entirety. Other human
antibodies can be obtained commercially from, for example, Abgenix,
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).
[0764] Completely human antibodies that recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al. (1994) Biotechnology 12:899-903). Human antibodies can also
be produced using various techniques known in the art, including
phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Quan,
M. P. and Carter, P. 2002. The rise of monoclonal antibodies as
therapeutics. In Anti-IgE and Allergic Disease, Jardieu, P. M. and
Fick Jr., R. B, eds., Marcel Dekker, New York, N.Y., Chapter 20,
pp. 427-469).
[0765] In other embodiments, the antibody is a fusion protein of an
antibody, or a functionally active fragment thereof, for example in
which the antibody is fused via a covalent bond (e.g., a peptide
bond), at either the N-terminus or the C-terminus to an amino acid
sequence of another protein (or portion thereof, preferably at
least 10, 20 or 50 amino acid portion of the protein) that is not
the antibody. Preferably, the antibody or fragment thereof is
covalently linked to the other protein at the N-terminus of the
constant domain.
[0766] Antibodies include analogs and derivatives that are either
modified, i.e, by the covalent attachment of any type of molecule
as long as such covalent attachment permits the antibody to retain
its antigen binding immunospecificity. For example, but not by way
of limitation, the derivatives and analogs of the antibodies
include those that have been further modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular antibody unit or other protein,
etc. Any of numerous chemical modifications can be carried out by
known techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis in the
presence of tunicamycin, etc. Additionally, the analog or
derivative can contain one or more unnatural amino acids.
[0767] The antibodies include antibodies having modifications
(e.g., substitutions, deletions or additions) in amino acid
residues that interact with Fc receptors. In particular, antibodies
include antibodies having modifications in amino acid residues
identified as involved in the interaction between the anti-Fc
domain and the FcRn receptor (see, e.g., International Publication
No. WO 97/34631, which is incorporated herein by reference in its
entirety). Antibodies immunospecific for a cancer cell antigen can
be obtained commercially, for example, from Genentech (San
Francisco, Calif.) or produced by any method known to one of skill
in the art such as, e.g., chemical synthesis or recombinant
expression techniques. The nucleotide sequence encoding antibodies
immunospecific for a cancer cell antigen can be obtained, e.g.,
from the GenBank database or a database like it, the literature
publications, or by routine cloning and sequencing.
[0768] In a specific embodiment, known antibodies for the treatment
or prevention of cancer can be used. Antibodies immunospecific for
a cancer cell antigen can be obtained commercially or produced by
any method known to one of skill in the art such as, e.g.,
recombinant expression techniques. The nucleotide sequence encoding
antibodies immunospecific for a cancer cell antigen can be
obtained, e.g., from the GenBank database or a database like it,
the literature publications, or by routine cloning and sequencing.
Examples of antibodies available for the treatment of cancer
include, but are not limited to, humanized anti-HER2 monoclonal
antibody, HERCEPTIN.RTM. (trastuzumab; Genentech) for the treatment
of patients with metastatic breast cancer; RITUXAN.RTM. (rituximab;
Genentech) which is a chimeric anti-CD20 monoclonal antibody for
the treatment of patients with non-Hodgkin's lymphoma; OvaRex
(AltaRex Corporation, MA) which is a murine antibody for the
treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is
a murine IgG.sub.2a antibody for the treatment of colorectal
cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) which is an
anti-EGFR IgG chimeric antibody for the treatment of epidermal
growth factor positive cancers, such as head and neck cancer;
Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the
treatment of sarcoma; Campath I/H (Leukosite, MA) which is a
humanized IgG.sub.1 antibody for the treatment of chronic
lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc.,
CA) which is a humanized anti-CD33 IgG antibody for the treatment
of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc.,
NJ) which is a humanized anti-CD22 IgG antibody for the treatment
of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc.,
CA) which is a humanized anti-HLA-DR antibody for the treatment of
non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a
radiolabeled murine anti-HLA-Dr10 antibody for the treatment of
non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a
humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or
non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA) which is an
anti-VEGF humanized antibody for the treatment of lung and
colorectal cancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen,
CA) which is an anti-CD22 antibody for the treatment of
non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ) which is a
humanized anti-CEA antibody for the treatment of colorectal
cancer.
[0769] Other antibodies useful in the treatment of cancer include,
but are not limited to, antibodies against the following antigens:
CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6
(carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha
fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline
phosphatase (carcinomas), prostate specific antigen (prostate),
prostatic acid phosphatase (prostate), epidermal growth factor
(carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3
(carcinomas), MAGE -4 (carcinomas), anti-transferrin receptor
(carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA
(colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate),
IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's
lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human
chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40
(lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma),
and Neu oncogene product (carcinomas). Some specific, useful
antibodies include, but are not limited to, BR96 mAb (Trail, P. A.,
Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J.,
Casazza, A. M., Firestone, R. A., Hellstrom, I., Hellstrom, K. E.,
"Cure of Xenografted Human Carcinomas by BR96-Doxorubicin
Immunoconjugates" Science 1993, 261, 212-215), BR64 (Trail, P A,
Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M.
E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M.,
Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R.
S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B.,
Hellstrom, K. E., and Hellstrom, I. "Effect of Linker Variation on
the Stability, Potency, and Efficacy of Carcinoma-reactive
BR64--Doxorubicin Immunoconjugates" Cancer Research 1997, 57,
100-105, mAbs against the CD40 antigen, such as S2C6 mAb
(Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and
Wahl, A. F. "Agonistic properties and in vivo antitumor activity of
the anti-CD-40 antibody, SGN-14" Cancer Res. 2000, 60, 3225-3231),
mAbs against the CD70 antigen, such as 1F6 mAb and 2F2 mAb, and
mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen,
K. J., Cheng, L., Avila, D., and Podack, E. R. "Functional effects
of CD30 on a large granular lymphoma cell line YT" J. Immunol.,
151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res.
62(13):3736-42). Many other internalizing antibodies that bind to
tumor associated antigens can be used and have been reviewed
(Franke, A. E., Sievers, E. L., and Scheinberg, D. A., "Cell
surface receptor-targeted therapy of acute myeloid leukemia: a
review" Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L.,
"Monoclonal antibody treatment of solid tumors: a coming of age"
Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S.,
Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
[0770] In certain embodiments, the antibody is not Trastuzumab
(full length, humanized anti-HER2 (MW 145167)),
HerceptinF(ab').sub.2 (derived from anti-HER2 enzymatically (MW
100000)), 4D5 (full-length, murine antiHER2, from hybridoma),
rhu4D5 (transiently expressed, full-length humanized antibody),
rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8
(full-length, murine antiHER2, with mutated FcRn binding domain),
or Hg ("Hingeless" full-length humanized 4D5, with heavy chain
hinge cysteines mutated to serines. Expressed in E. coli (therefore
non-glycosylated)).
[0771] In another specific embodiment, known antibodies for the
treatment or prevention of an autoimmune disease are used in
accordance with the compositions and methods of the invention.
Antibodies immunospecific for an antigen of a cell that is
responsible for producing autoimmune antibodies can be obtained
from any organization (e.g., a university scientist or a company)
or produced by any method known to one of skill in the art such as,
e.g., chemical synthesis or recombinant expression techniques. In
another embodiment, useful antibodies are immunospecific for the
treatment of autoimmune diseases include, but are not limited to,
Anti-Nuclear Antibody; Anti-ds DNA; Anti-ss DNA, Anti-Cardiolipin
Antibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SM
Antibody; Anti-Mitochondrial Antibody; Thyroid Antibody; Microsomal
Antibody; Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo;
Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital
Cells Antibody; Anti-Histones; Anti-RNP; C-ANCA; P-ANCA; Anti
centromere; Anti-Fibrillarin, and Anti-GBM Antibody.
[0772] In certain embodiments, useful antibodies can bind to both a
receptor or a receptor complex expressed on an activated
lymphocyte. The receptor or receptor complex can comprise an
immunoglobulin gene superfamily member, a TNF receptor superfamily
member, an integrin, a cytokine receptor, a chemokine receptor, a
major histocompatibility protein, a lectin, or a complement control
protein. Non-limiting examples of suitable immunoglobulin
superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79,
CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of
suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas,
CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,
osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and
APO-3. Non-limiting examples of suitable integrins are CD11a,
CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e,
CD49f, CD103, and CD104. Non-limiting examples of suitable lectins
are C-type, S-type, and I-type lectin.
[0773] In one embodiment, the Ligand binds to an activated
lymphocyte that is associated with an autoimmune disease.
[0774] In another specific embodiment, useful Ligands
immunospecific for a viral or a microbial antigen are monoclonal
antibodies. The antibodies may be chimeric, humanized or human
monoclonal antibodies. As used herein, the term "viral antigen"
includes, but is not limited to, any viral peptide, polypeptide
protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza
virus neuraminidase, influenza virus hemagglutinin, HTLV tax,
herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and
hepatitis B surface antigen) that is capable of eliciting an immune
response. As used herein, the term "microbial antigen" includes,
but is not limited to, any microbial peptide, polypeptide, protein,
saccharide, polysaccharide, or lipid molecule (e.g., a bacterial,
fungi, pathogenic protozoa, or yeast polypeptide including, e.g.,
LPS and capsular polysaccharide 5/8) that is capable of eliciting
an immune response.
[0775] Antibodies immunospecific for a viral or microbial antigen
can be obtained commercially, for example, from BD Biosciences (San
Francisco, Calif.), Chemicon International, Inc. (Temecula,
Calif.), or Vector Laboratories, Inc. (Burlingame, Calif.) or
produced by any method known to one of skill in the art such as,
e.g., chemical synthesis or recombinant expression techniques. The
nucleotide sequence encoding antibodies that are immunospecific for
a viral or microbial antigen can be obtained, e.g., from the
GenBank database or a database like it, literature publications, or
by routine cloning and sequencing.
[0776] In a specific embodiment, useful Ligands are those that are
useful for the treatment or prevention of viral or microbial
infection in accordance with the methods disclosed herein. Examples
of antibodies available useful for the treatment of viral infection
or microbial infection include, but are not limited to, SYNAGIS
(MedImmune, Inc., MD) which is a humanized anti-respiratory
syncytial virus (RSV) monoclonal antibody useful for the treatment
of patients with RSV infection; PRO542 (Progenics) which is a CD4
fusion antibody useful for the treatment of HIV infection; OSTAVIR
(Protein Design Labs, Inc., CA) which is a human antibody useful
for the treatment of hepatitis B virus; PROTOVIR (Protein Design
Labs, Inc., CA) which is a humanized IgG.sub.1 antibody useful for
the treatment of cytomegalovirus (CMV); and anti-LPS
antibodies.
[0777] Other antibodies useful in the treatment of infectious
diseases include, but are not limited to, antibodies against the
antigens from pathogenic strains of bacteria (Streptococcus
pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria
meningitidis, Corynebacterium diphtheriae, Clostridium botulinum,
Clostridium perfringens, Clostridium tetani, Hemophilus influenzae,
Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella
rhinoscleromotis, Staphylococc aureus, Vibrio colerae, Escherichia
coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus,
Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia
enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,
Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Salmonella typhimurium, Treponema pallidum, Treponema pertenue,
Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,
Leptospira icterohemorrhagiae, Mycobacterium tuberculosis,
Pneumocystis carinii, Francisella tularensis, Brucella abortus,
Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia
prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic
fungi (Coccidioides immitis, Aspergillus fumigatus, Candida
albicans, Blastomyces dermatitidis, Cryptococcus neoformans,
Histoplasma capsulatum); protozoa (Entomoeba histolytica,
Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis,
Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma
rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania
tropica, Leishmania braziliensis, Pneumocystis pneumonia,
Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or
Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris
lumbricoides, Trichinella spiralis, Strongyloides stercoralis,
Schistosoma japonicum, Schistosoma mansoni, Schistosoma
haematobium, and hookworms).
[0778] Other antibodies useful in this invention for treatment of
viral disease include, but are not limited to, antibodies against
antigens of pathogenic viruses, including as examples and not by
limitation: Poxyiridae, Herpesviridae, Herpes Simplex virus 1,
Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae,
Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza
viruses, parainfluenza viruses, mumps, measles, respiratory
syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,
Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis
E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae,
Rotoviridae, and Human Immunodeficiency Virus.
[0779] In attempts to discover effective cellular targets for
cancer diagnosis and therapy, researchers have sought to identify
transmembrane or otherwise tumor-associated polypeptides that are
specifically expressed on the surface of one or more particular
type(s) of cancer cell as compared to on one or more normal
non-cancerous cell(s). Often, such tumor-associated polypeptides
are more abundantly expressed on the surface of the cancer cells as
compared to on the surface of the non-cancerous cells. The
identification of such tumor-associated cell surface antigen
polypeptides has given rise to the ability to specifically target
cancer cells for destruction via antibody-based therapies.
[0780] Antibodies which comprise Ab in Formula Ic antibody drug
conjugates (ADC) and which may be useful in the treatment of cancer
include, but are not limited to, antibodies against
tumor-associated antigens (TAA). Such tumor-associated antigens are
known in the art, and can prepared for use in generating antibodies
using methods and information which are well known in the art.
Examples of TAA include (1)-(35), but are not limited to TAA
(1)-(35) listed below. For convenience, information relating to
these antigens, all of which are known in the art, is listed below
and includes names, alternative names, Genbank accession numbers
and primary reference(s). Tumor-associated antigens targeted by
antibodies include all amino acid sequence variants and isoforms
possessing at least about 70%, 80%, 85%, 90%, or 95% sequence
identity relative to the sequences identified in the corresponding
sequences listed (SEQ ID NOS: 1-35) or the sequences identified in
the cited references. In some embodiments, TAA having amino acid
sequence variants exhibit substantially the same biological
properties or characteristics as a TAA having the sequence found in
the corresponding sequences listed (SEQ ID NOS: 1-35). For example,
a TAA having a variant sequence generally is able to bind
specifically to an antibody that binds specifically to the TAA with
the corresponding sequence listed. The sequences and disclosure
specifically recited herein are expressly incorporated by
reference.
[0781] Tumor-Associated Antigens (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank
accession no. NM.sub.--001203, ten Dijke, P., et al. Science 264
(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997));
WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1
(Page 38-39); WO2002102235 (claim 13; Page 296); WO2003055443 (Page
91-92); WO200299122 (Example 2; Page 528-530); WO2003029421 (claim
6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; Page
183); WO200254940 (Page 100-101); WO200259377(Page 349-350);
WO200230268 (claim 27; Page 376); WO200148204 (Example; FIG. 4)
NP.sub.--001194 bone morphogenetic protein receptor, type
IB/pid=NP.sub.--001194.1-Cross-references: MIM:603248;
NP.sub.--001194.1; NM.sub.--001203.sub.--1
TABLE-US-00004 502 aa (SEQ ID NO: 1)
MLLRSAGKLNVGTKKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTDGYCFTMIEED
DSGLPVVTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERNECNKDLHPTLPPLKNRDFVD
GPIHHRALLISVTVCSLLLVLIILFCYFRYKRQETRPRYSIGLEQDETYIPPGESLRDLI
EQSQSSGSGSGLPLLVQRTIAKQIQMVKQIGKGRYGEVWMGKWRGEKVAVKVFFTTEEAS
WFRETEIYQTVLMRHENILGFIAADIKGTGSWTQLYLITDYHENGSLYDYLKSTTLDAKS
MLKLAYSSVSGLCHLHTEIFSTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVKFISD
TNEVDIPPNTRVGTKRYMPPEVLDESLNRNHFQSYIMADMYSFGLILWEVARRCVSGGIV
EEYQLPYHDLVPSDPSYEDMREIVCIKKLRPSFPNRWSSDECLRQMGKLMTECWAHNPAS
RLTALRVKKTLAKMSESQDIKL
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.--003486);
Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395
(6699):288-291 (1998), Gaugitsch, H. W., et al. (1992) J. Biol.
Chem. 267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842
(Example IV); WO2003042661 (claim 12); WO2003016475 (claim 1);
WO200278524 (Example 2); WO200299074 (claim 19; Page 127-129);
WO200286443 (claim 27; Pages 222, 393); WO2003003906 (claim 10;
Page 293); WO200264798 (claim 33; Page 93-95); WO200014228 (claim
5; Page 133-136); US2003224454 (FIG. 3); WO2003025138 (claim 12;
Page 150); NP.sub.--003477 solute carrier family 7 (cationic amino
acid transporter, y+ system), member 5/pid=NP.sub.--003477.3-Homo
sapiens
Cross-references: MIM:600182; NP.sub.--003477.3; NM.sub.--015923;
NM.sub.--003486.sub.--1
TABLE-US-00005 [0782] 507 aa (SEQ ID NO: 2)
MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNITLLNGVAIIV
GTIIGSGIFVTPTGVLKEAGSPGLALVVWAACGVFSIVGALCYAELGTTISKSGGDYAYM
LEVYGSLPAFLKLWIELLIIRPSSQYIVALVFATYLLKPLFPTCPVPEEAAKLVACLCVL
LLTAVNCYSVKAATRVQDAFAAAKLLALALIILLGFVQIGKGVVSNLDPNFSFEGTKLDV
GNIVLALYSGLFAYGGWNYLNFVTEEMINPYRNLPLAIIISLPIVTLVYVLTNLAYFTTL
STEQMLSSEAVAVDFGNYHLGVMSWIIPVFVGLSCFGSVNGSLFTSSRLFFVGSREGHLP
SILSMIHPQLLTPVPSLVFTCVMTLLYAFSKDIFSVINFFSFFNWLCVALAIIGMIWLRH
RKPELERPIKVNLALPVFFILACLFLIAVSFWKTPVECGIGFTIILSGLPVYFFGVWWKN
KPKWLLQGIFSTTVLCQKLMQVVPQET
(3) STEAP1 (six transmembrane epithelial antigen of prostate,
Genbank accession no. NM.sub.--012449 Cancer Res. 61 (15),
5857-5860 (2001), Hubert, R. S., et al. (1999) Proc. Natl. Acad.
Sci. USA. 96 (25):14523-14528); WO2004065577 (claim 6);
WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (claim
2); WO2003042661 (claim 12); US2003157089 (Example 5); US2003185830
(Example 5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page
618-619); WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173,
Example 2; FIG. 2A); NP.sub.--036581 six transmembrane epithelial
antigen of the prostate
Cross-references: MIM:604415; NP.sub.--036581.1;
NM.sub.--012449.sub.--1
TABLE-US-00006 [0783] 339 aa (SEQ ID NO 3)
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
VSDSLTWREFHYIQSKLGIVSLLLGTTHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIV
VLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486 J. Biol.
Chem. 276 (29):27371-27375 (2001)); WO2004045553 (claim 14);
WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; Page
116-121); US2003124140 (Example 16); US2003091580 (claim 6);
WO200206317 (claim 6; Page 400-408);
Cross-references: GI:34501467; AAK74120.3; AF361486.sub.--1
TABLE-US-00007 [0784] 6995 aa (SEQ ID NO: 4)
PVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEAMQPSTHTAVT
NVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSL
TSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDIS
TEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHS
EMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALL
TLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVI
YKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREIS
TSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRIS
TPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSR
GPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKT
TDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYS
SYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIH
SATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTES
TEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGEVPWLS
HPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSE
PETSSPPNLSSTSAEILATTEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSM
GITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPT
GATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPS
GATSQGTFTLDSSSTASWPGTHSATTQRFPRSVVTTPMSRGPEDVSWPSPLSVEKNSPPS
SLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNI
TSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSS
IRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPE
VSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQ
VTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLP
LTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIP
ATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVEDTTV
FTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLFGVPTSATTEVSMTEI
MSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLA
TTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSPFVEKTSSSSSLLSLPVTTSPS
VSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTE
KIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFET
TGFEAEPFSHLTSGLRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDW
PPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETIS
TGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSS
STFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSS
PILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQT
STSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASMQ
MASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSR
SGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGA
DVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTI
PRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPT
LTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNS
PGEPATTVSLVTHSAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEV
PGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVV
TSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVAS
SRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNS
TSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIP
ILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTFLVTSSRAVTSTTIPTLTISS
DEPETTTSLVTHSEAKMISAIPTLGVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPET
IDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPS
TVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVTHP
AVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTD
TSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPDASKMLTSLVISSGTDSTTT
FPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVS
TTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTIPN
FSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPG
EPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPD
ATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALL
STHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVD
LSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLT
VSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPP
KTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLA
SESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMV
PFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPE
KDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHR
SSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRK
FNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLN
REQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSS
LSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVG
PLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYT
LDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITN
LKYEEDMHRPGSRKFNTTERVLQTLVGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDA
ICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPG
TSTVDLGSGTPSSLPSPTSATAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQ
SLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELS
QLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGP
LLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLL
RPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGF
THRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPG
SRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSP
GLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGT
PSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNSSV
GPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPY
SLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTIT
NLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVD
TICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTP
GTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQ
GLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELS
QLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGP
LLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLL
RPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGF
THRNFVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPG
SRKFNTTERVLQGLLRPLFKNTSIGPLYSSCRLTLLRPEKDKAATRVDAICTHHPDPQSP
GLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGI
PPSLPETTATGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSV
GPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPY
TLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTII
NLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVD
AVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTP
DTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQ
GLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELS
QLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASP
LLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTLL
RPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGF
THRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGS
RKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPG
LDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTI
NNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRV
DLLCTYLQPLSGPGLPIKQVFHELSQQTRGITRLGPYSLDKDSLYLNGYNEPGPDEPPTT
PKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRP
LFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHG
VTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQD
KVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHW
LGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRN
IEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFL
RMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGLITCLIC
GVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823 Yamaguchi, N., et
al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci.
USA. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. USA. 93
(1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995));
WO2003101283 (claim 14); (WO2002102235 (claim 13; Page 287-288);
WO2002101075 (claim 4; Page 308-309); WO200271928 (Page 320-321);
WO9410312 (Page 52-57);
Cross-references: MIM:601051; NP.sub.--005814.2;
NM.sub.--005823.sub.--1
TABLE-US-00008 [0785] 622 aa (SEQ ID NO: 5)
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS
LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL
DLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA
DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTW
SVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT
ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELY
PQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVK
GRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKA
RLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQ
KLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGT
PCLLGPGPVLTVLALLLASTLA
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424, J. Biol.
Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999),
Feild, J. A., et al. (1999) Biochem. Biophys. Res. Commun. 258
(3):578-582); WO2004022778 (claim 2); EP1394274 (Example 11);
WO2002102235 (claim 13; Page 326); EP875569 (claim 1; Page 17-19);
WO200157188 (claim 20; Page 329); WO2004032842 (Example IV);
WO200175177 (claim 24; Page 139-140);
Cross-references: MIM:604217; NP.sub.--006415.1;
NM.sub.--006424.sub.--1
TABLE-US-00009 [0786] 690 aa (SEQ ID NO: 6)
MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKIEL
LPSYSTATLIDEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGR
LILLLGFLYFFVCSLDILSSAFQLVGGKMAGQFFSNSSIMSNPLLGLVIG
VLVTVLVQSSSTSTSIVVSMVSSSLLTVRAAIPIIMGANIGTSITNTIVA
LMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHYLEIITQLIVE
SFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVK
IWCKTFTNKTQINVTVPSTANCTSPSLCWTDGIQNWTMKNVTYKENIAKC
QHIFVNFHLPDLAVGTILLILSLLVLCGCLIMIVKILGSVLKGQVATVIK
KTINTDFPFPFAWLTGYLAILVGAGMTFIVQSSSVFTSALTPLIGIGVIT
IERAYPLTLGSNIGTTTTAILAALASPGNALRSSLQIALCHFFFNISGIL
LWYPIPFTRLPIRMAKGLGNISAKYRWFAVFYLIIFFFLIPLTVFGLSLA
GWRVLVGVGVPVVFIIILVLCLRLLQSRCPRVLPKKLQNWNFLPLWMRSL
KPWDAVVSKFTGCFQMRCCYCCRVCCRACCLLCGCPKCCRCSKCCEDLEE
AQEGQDVPVKAPETFDNITISREAQGEVPASDSKTECTAL
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878, Nagase T.,
et al. (2000) DNA Res. 7 (2):143-150); WO2004000997 (claim 1);
WO2003003984 (claim 1); WO200206339 (claim 1; Page 50); WO200188133
(claim 1; Page 41-43, 48-58); WO2003054152 (claim 20); WO2003101400
(claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;
HGNC:10737;
TABLE-US-00010 1093 aa (SEQ ID NO: 7)
MVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCALSKHPTVAF
EDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQ
ATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMC
TSRQVGNLSRTTEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGR
DPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAYFFLRENAVEH
DCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNE
LQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQEN
PRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPV
TPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRS
LHGCYLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAA
YRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACPVRNVTR
DGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHI
ANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSR
EERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGC
GVEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLAD
PHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAA
WGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDCNPQACP
VRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEAL
CATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRP
CPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLS
CQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIP
DDRANFYPLQQTNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
US2003129192 (claim 2); US2004044180 (claim 12); US2004044179
(claim 11); US2003096961 (claim 11); US2003232056 (Example 5);
WO2003105758 (claim 12); US2003206918 (Example 5); EP1347046 (claim
1); WO2003025148 (claim 20); Cross-references: GI:37182378;
AAQ88991.1; AY358628.sub.--1
TABLE-US-00011 141 aa (SEQ ID NO: 8)
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAGYQSFCSPGKLNSVCISC
CNTPLCNGPRPKKRGSSASALRPGLRTTILFLKLALFSAHC
(9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463); Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177,
34-39, 1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178,
248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992;
Arai H., et al. J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A.,
Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178, 656-663,
1991; Elshourbagy N. A., et al. J. Biol. Chem. 268, 3873-3879,
1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1-s4,
1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg R. L.,
et al. Proc. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; Bourgeois
C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto
Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij J. B., et
al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al.
Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al.
Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4,
2407-2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351-354,
1996; Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra
R. M. W., et al. Nat. Genet. 12, 445-447, 1996; Svensson P. J., et
al. Hum. Genet. 103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7,
115-124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, 198-206;
WO2004045516 (claim 1); WO2004048938 (Example 2); WO2004040000
(claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1);
WO2003016475 (claim 1); WO200261087 (FIG. 1); WO2003016494 (FIG.
6); WO2003025138 (claim 12; Page 144); WO200198351 (claim 1; Page
124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; Page
297-299); US2003109676; U56518404 (FIG. 3); U.S. Pat. No. 5,773,223
(Claim 1a; Col 31-34); WO2004001004;
TABLE-US-00012 442 aa (SEQ ID NO: 9)
MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPT
KTLWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETF
KYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLH
IVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRY
RAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGFDIITMDYKGS
YLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEM
LRKKSGMQIALNDHLKQRREVAKTVFCLVLVFALCWLPLHLSRILKLTLY
NQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSC
LCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFRSSNKYSSS
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763); WO2003104275 (claim 1);
WO2004046342 (Example 2); WO2003042661 (claim 12); WO2003083074
(claim 14; Page 61); WO2003018621 (claim 1); WO2003024392 (claim 2;
FIG. 93); WO200166689 (Example 6);
Cross-references: LocusID:54894; NP.sub.--060233.2;
NM.sub.--017763.sub.--1
TABLE-US-00013 [0787] 783 aa (SEQ ID NO: 10)
MSGGHQLQLAALWPWLLMATLQAGFGRTGLVLAAAVESERSAEQKAIIRV
IPLKMDPTGKLNLTLEGVFAGVAEITPAEGKLMQSHPLYLCNASDDDNLE
PGFISIVKLESPRRAPRPCLSLASKARMAGERGASAVLFDITEDRAAAEQ
LQQPLGLTWPVVLIWGNDAEKLMEFVYKNQKAHVRIELKEPPAWPDYDVW
ILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQA
SCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVD
PWLHQHRTCPLCVFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAH
YHLPAAYLLGPSRSAVARPPRPGPFLPSQEPGMGPRHHRFPRAAHPRAPG
EQQRLAGAQHPYAQGWGMSHLQSTSQHPAACPVPLRRARPPDSSGSGESY
CTERSGYLADGPASDSSSGPCHGSSSDSVVNCTDISLQGVHGSSSTFCSS
LSSDFDPLVYCSPKGDPQRVDMQPSVTSRPRSLDSVVPTGETQVSSHVHY
HRHRHHHYKKRFQWHGRKPGPETGVPQSRPPIPRTQPQPEPPSPDQQVTG
SNSAAPSGRLSNPQCPRALPEPAPGPVDASSICPSTSSLFNLQKSSLSAR
HPQRKRRGGPSEPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHPLI
CGPPGLDKRLLPETPGPCYSNSQPVWLCLTPRQPLEPHPPGEGPSEWSSD
TAEGRPCPYPHCQVLSAQPGSEEELEELCEQAV
(11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no. AF455138,
Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397
(claim 1; FIG. 1); WO200272596 (claim 13; Page 54-55); WO200172962
(claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim
16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612
(claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429
(claim 12; FIG. 10);
Cross-references: GI:22655488; AAN04080.1; AF455138.sub.--1
TABLE-US-00014 [0788] 490 aa (SEQ ID NO: 11)
MESISMMGSPKSLSETVLPNGINGIKDARKVTVGVIGSGDFAKSLTIRLI
RCGYHVVIGSRNPKFASEFFPHVVDVTHHEDALTKTNIIFVAIHREHYTS
LWDLRHLLVGKILIDVSNNMRINQYPESNAEYLASLFPDSLIVKGFNVVS
AWALQLGPKDASRQVYICSNNIQARQQVIELARQLNFIPIDLGSLSSARE
IENLPLRLFTLWRGPVVVAISLATFFFLYSFVRDVIHPYARNQQSDFYKI
PIEIVNKTLPIVAITLLSLVYLAGLLAAAYQLYYGTKYRRFPPWLETWLQ
CRKQLGLLSFFFAMVHVAYSLCLPMRRSERYLFLNMAYQQVHANIENSWN
EEEVWRIEMYISFGIMSLGLLSLLAVTSIPSVSNALNWREFSFIQSTLGY
VALLISTFHVLIYGWKRAFEEEYYRFYTPPNFVLALVLPSIVILGKIILF
LPCISQKLKRIKKGWEKSQFLEEGIGGTIPHVSPERVTVM
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor
potential cation channel, subfamily M, member 4, Genbank accession
no. NM.sub.--017636 Xu, X. Z., et al. Proc. Natl. Acad. Sci. USA.
98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol.
Chem. 278 (33):30813-30820 (2003)); US2003143557 (claim 4);
WO200040614 (claim 14; Page 100-103); WO200210382 (claim 1; FIG.
9A); WO2003042661 (claim 12); WO200230268 (claim 27; Page 391);
US2003219806 (claim 4); WO200162794 (claim 14; FIG. 1A-D);
Cross-references: MIM:606936; NP.sub.--060106.2;
NM.sub.--017636.sub.--1
TABLE-US-00015 [0789] 1214 aa (SEQ ID NO: 12)
MVVPEKEQSWIPKIFKKKTCTTFIVDSTDPGGTLCQCGRPRTAHPAVAME
DAFGAAVVTVWDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDRTDPA
AVYSLVTRTWGFRAPNLVVSVLGGSGGPVLQTWLQDLLRRGLVRAAQSTG
AWIVTGGLHTGIGRHVGVAVRDHQMASTGGTKVVAMGVAPWGVVRNRDTL
INPKGSFPARYRWRGDPEDGVQFPLDYNYSAFFLVDDGTHGCLGGENRFR
LRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMLTRIENATQAQLPCLL
VAGSGGAADCLAETLEDTLAPGSGGARQGEARDRIRRFFPKGDLEVLQAQ
VERIMTRKELLTVYSSEDGSEEFETIVLKALVKACGSSEASAYLDELRLA
VAWNRVDIAQSELFRGDIQWRSFHLEASLMDALLNDRPEFVRLLISHGLS
LGHFLTPMRLAQLYSAAPSNSLIRNLLDQASHSAGTKAPALKGGAAELRP
PDVGHVLRMLLGKMCAPRYPSGGAWDPHPGQGFGESMYLLSDKATSPLSL
DAGLGQAPWSDLLLWALLLNRAQMAMYFWEMGSNAVSSALGACLLLRVMA
RLEPDAEEAARRKDLAFKFEGMGVDLFGECYRSSEVRAARLLLRRCPLWG
DATCLQLAMQADARAFFAQDGVQSLLTQKWWGDMASTTPIWALVLAFFCP
PLIYTRLITFRKSEEEPTREELEFDMDSVINGEGPVGTADPAEKTPLGVP
RQSGRPGCCGGRCGGRRCLRRWFHFWGAPVTIFMGNVVSYLLFLLLFSRV
LLVDFQPAPPGSLELLLYFWAFTLLCEELRQGLSGGGGSLASGGPGPGHA
SLSQRLRLYLADSWNQCDLVALTCFLLGVGCRLTPGLYHLGRTVLCIDFM
VFTVRLLHIFTVNKQLGPKIVIVSKMMKDVFFFLFFLGVWLVAYGVATEG
LLRPRDSDFPSILRRVFYRPYLQIFGQIPQEDMDVALMEHSNCSSEPGFW
AHPPGAQAGTCVSQYANWLVVLLLVIFLLVANILLVNLLIAMFSYTFGKV
QGNSDLYWKAQRYRLIREFHSRPALAPPFIVISHLRLLLRQLCRRPRSPQ
PSSPALEHFRVYLSKEAERKLLTWESVHKENFLLARARDKRESDSERLKR
TSQKVDLALKQLGHIREYEQRLKVLEREVQQCSRVLGWVAEALSRSALLP
PGGPPPPDLPGSKD
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived
growth factor, Genbank accession no. NP.sub.--003203 or
NM.sub.--003212, Ciccodicola, A., et al. EMBO J. 8 (7):1987-1991
(1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003224411
(claim 1); WO2003083041 (Example 1); WO2003034984 (claim 12);
WO200288170 (claim 2; Page 52-53); WO2003024392 (claim 2; FIG. 58);
WO200216413 (claim 1; Page 94-95, 105); WO200222808 (claim 2; FIG.
1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18); U.S. Pat. No.
5,792,616 (FIG. 2);
Cross-references: MIM:187395; NP.sub.--003203.1;
NM.sub.--003212.sub.--1
TABLE-US-00016 [0790] 188 aa (SEQ ID NO: 13)
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS
IWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr
virus receptor) or Hs.73792 Genbank accession no. M26004, Fujisaku
et al. (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al.
J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl.
Acad. Sci. USA. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol.
35, 1025-1031, 1998; Weis J. J., et al. Proc. Natl. Acad. Sci. USA.
83, 5639-5643, 1986; Sinha S. K., et al. (1993) J. Immunol. 150,
5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1);
WO2003062401 (claim 9); WO2004045520 (Example 4); WO9102536 (FIG.
9.1-9.9); WO2004020595 (claim 1); Accession: P20023; Q13866;
Q14212; EMBL; M26004; AAA35786.1.
TABLE-US-00017 1033 aa (SEQ ID NO: 14)
MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYS
CSGTFRLIGEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGY
KIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQANNMWGPTRLPTCVSVF
PLECPALPMIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSS
GKWSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANFFCDEGYRLQG
PPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPILNGRHIGNSLANVSYGSI
VTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTWSGPAPRCELSTSAVQ
CPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWE
PSAPVCEKECQAPPNILNGQKEDRHMVRFDPGTSIKYSCNPGYVLVGEES
IQCTSEGVWTPPVPQCKVAACEATGRQLLTKPQHQFVRPDVNSSCGEGYK
LSGSVYQECQGTIPWFMEIRLCKEITCPPPPVIYNGAHTGSSLEDFPYGT
TVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAV
QCSHVHIANGYKISGKEAPYFYNDTVTFKCYSGFTLKGSSQIRCKADNTW
DPEIPVCEKETCQHVRQSLQELPAGSRVELVNTSCQDGYQLTGHAYQMCQ
DAENGIWFKKIPLCKVIHCHPPPVIVNGKHTGMMAENFLYGNEVSYECDQ
GFYLLGEKKLQCRSDSKGHGSWSGPSPQCLRSPPVTRCPNPEVKHGYKLN
KTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIG
CPPPPKTPNGNHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTW
SQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGS
PQSQCQSDHQWNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISK
HRERNYYTDTSQKEAFHLEAREVYSVDPYNPAS
(15) CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated
beta), B29, Genbank accession no. NM.sub.--000626 or 11038674,
Proc. Natl. Acad. Sci. USA. (2003) 100 (7):4126-4131, Blood (2002)
100 (9):3068-3076, Muller et al. (1992) Eur. J. Immunol. 22
(6):1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768,
US2004101874 (claim 1, page 102); WO2003062401 (claim 9);
WO200278524 (Example 2); US2002150573 (claim 5, page 15); U.S. Pat.
No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO
99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B);
WO200055351 (claim 11, pages 1145-1146); Cross-references:
MIM:147245; NP.sub.--000617.1; NM.sub.--000626.sub.--1
TABLE-US-00018 229 aa (SEQ ID NO: 15)
MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSP
RFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQ
NESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQ
LKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLD
IDQTATYEDIVTLRTGEVKWSVGEHPGQE
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase
anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no.
NM.sub.--030764, Genome Res. 13 (10):2265-2270 (2003),
Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002),
Proc. Natl. Acad. Sci. USA. 98 (17):9772-9777 (2001), Xu, M. J., et
al. (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775;
WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; FIG.
18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25);
Cross-references: MIM:606509; NP.sub.--110391.2;
NM030764.sub.--1
TABLE-US-00019 [0791] 508 aa (SEQ ID NO: 16)
MLLWSLLVIFDAVTEQADSLTLVAPSSVFEGDSIVLKCQGEQNWKIQKMA
YHKDNKELSVFKKFSDFLIQSAVLSDSGNYFCSTKGQLFLWDKTSNIVKI
KVQELFQRPVLTASSFQPIEGGPVSLKCETRLSPQRLDVQLQFCFFRENQ
VLGSGWSSSPELQISAVWSEDTGSYWCKAETVTHRIRKQSLQSQIHVQRI
PISNVSLEIRAPGGQVTEGQKLILLCSVAGGTGNVTFSWYREATGTSMGK
KTQRSLSAELEIPAVKESDAGKYYCRADNGHVPIQSKVVNIPVRIPVSRP
VLTLRSPGAQAAVGDLLELHCEALRGSPPILYQFYHEDVTLGNSSAPSGG
GASFNLSLTAEHSGNYSCEANNGLGAQCSEAVPVSISGPDGYRRDLMTAG
VLWGLFGVLGFTGVALLLYALFHKISGESSATNEPRGASRPNPQEFTYSS
PTPDMEELQPVYVNVGSVDVDVVYSQVWSMQQPESSANIRTLLENKDSQV IYSSVKKS
(17) HER2 (ErbB2, Genbank accession no. M11730, Coussens L., et al.
Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature
319, 230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. USA.
82, 6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol. 165,
869-880, 2004; Kuhns J. J., et al. J. Biol. Chem. 274, 36422-36427,
1999; Cho H.-S., et al. Nature 421, 756-760, 2003; Ehsani A., et
al. (1993) Genomics 15, 426-429; WO2004048938 (Example 2);
WO2004027049 (FIG. 1e; WO2004009622; WO2003081210; WO2003089904
(claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537
(claim 1); WO2003055439 (claim 29; FIG. 1A-B); WO2003025228 (claim
37; FIG. 5C); WO200222636 (Example 13; Page 95-107); WO200212341
(claim 68; FIG. 7); WO200213847 (Page 71-74); WO200214503 (Page
114-117); WO200153463 (claim 2; Page 41-46); WO200141787 (Page 15);
WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); U.S.
Pat. No. 5,869,445 (claim 3; Col 31-38); WO9630514 (claim 2; Page
56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709;
WO200100244 (Example 3; FIG. 4); Accession: PO4626; EMBL; M11767;
AAA35808.1. EMBL; M11761; AAA35808.1.
TABLE-US-00020 1255 aa (SEQ ID NO: 17)
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLY
QGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLR
IVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILK
GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK
GSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHS
DCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP
YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHL
REVRAVTSANIQEFAGCKKIFGSLAFLPESEDGDPASNTAPLQPEQLQVF
ETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI
SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRP
EDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGL
PREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC
PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASP
LTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPL
TPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPV
AIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQL
MPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARN
VLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT
HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTID
VYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL
DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSS
STRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQS
LPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPP
SPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ
GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLG LDVPV
(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al
Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res.
Commun. 150, 89-96, 1988; Strausberg R. L., et al. Proc. Natl.
Acad. Sci. USA. 99:16899-16903, 2002; WO2004063709; EP1439393
(claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661
(claim 12); WO200278524 (Example 2); WO200286443 (claim 27; Page
427); WO200260317 (claim 2);
Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL;
M18728;
TABLE-US-00021 [0792] 344 aa (SEQ ID NO: 18)
MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKE
VLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRET
IYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSIS
SNNSNPVEDKDAVAFTCEPEVQNTTYLWWVNGQSLPVSPRLQLSNGNMTL
TLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVPTISPSKANYR
PGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQ
AHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI
(19) MDP (DPEP1, Genbank accession no. BC017023, Proc. Natl. Acad.
Sci. USA. 99 (26):16899-16903 (2002)); WO2003016475 (claim 1);
WO200264798 (claim 33; Page 85-87); JP05003790 (FIG. 6-8);
WO9946284 (FIG. 9);
Cross-references: MIM:179780; AAH17023.1; BC017023.sub.--1
TABLE-US-00022 [0793] 411 aa (SEQ ID NO: 19)
MWSGWWLWPLVAVCTADFFRDEAERIMRDSPVIDGHNDLPWQLLDMFNNR
LQDERANLTTLAGTHTNIPKLRAGFVGGQFWSVYTPCDTQNKDAVRRTLE
QMDVVHRMCRMYPETFLYVTSSAGIRQAFREGKVASLIGVEGGHSIDSSL
GVLRALYQLGMRYLTLTHSCNTPWADNWLVDTGDSEPQSQGLSPFGQRVV
KELNRLGVLIDLAHVSVATMKATLQLSRAPVIFSHSSAYSVCASRRNVPD
DVLRLVKQTDSLVMVNFYNNYISCTNKANLSQVADHLDHIKEVAGARAVG
FGGDFDGVPRVPEGLEDVSKYPDLIAELLRRNWTEAEVKGALADNLLRVF
EAVEQASNLTQAPEEEPIPLDQLGGSCRTHYGYSSGASSLHRHWGLLLAS LAPLVLCLSLL
(20) IL20R.alpha. (IL20Ra, ZCYTOR7, Genbank accession no. AF
184971); Clark H. F., et al. Genome Res. 13, 2265-2270, 2003;
Mungall A. J., et al. Nature 425, 805-811, 2003; Blumberg H., et
al. Cell 104, 9-19, 2001; Dumoutier L., et al. J. Immunol. 167,
3545-3549, 2001; Parrish-Novak J., et al. J. Biol. Chem. 277,
47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry
42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172,
2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);
WO2003029262 (Page 74-75); WO2003002717 (claim 2; Page 63);
WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261
(Page 57-59); WO200146232 (Page 63-65); WO9837193 (claim 1; Page
55-59);
Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.
TABLE-US-00023 [0794] 553 aa (SEQ ID NO: 20)
MRAPGRPALRPLPLPPLLLLLLAAPWGRAVPCVSGGLPKPANITFLSINM
KNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAE
TSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKS
ISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTN
HTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEFKAK
IIFWYVLPISITVFLFSVMGYSIYRYIHVGKEKHPANLILIYGNEFDKRF
FVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDVSSLNDPQPSGNLRP
PQEEEEVKHLGYASHLMEIFCDSEENTEGTSFTQQESLSRTIPPDKTVIE
YEYDVRTTDICAGPEEQELSLQEEVSTQGTLLESQAALAVLGPQTLQYSY
TPQLQDLDPLAQEHTDSEEGPEEEPSTTLVDWDPQTGRLCIPSLSSFDQD
SEGCEPSEGDGLGEEGLLSRLYEEPAPDRPPGENETYLMQFMEEWGLYVQ MEN
(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053) Gary S.
C., et al. Gene 256, 139-147, 2000; Clark H. F., et al. Genome Res.
13, 2265-2270, 2003; Strausberg R. L., et al. Proc. Natl. Acad.
Sci. USA. 99, 16899-16903, 2002; US2003186372 (claim 11);
US2003186373 (claim 11); US2003119131 (claim 1; FIG. 52);
US2003119122 (claim 1; FIG. 52); US2003119126 (claim 1);
US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1);
US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52);
US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim
1);
TABLE-US-00024 911 aa (SEQ ID NO: 21)
MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLGGALTIPCHVH
YLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDV
SLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGVVFLYREGSARYAFSFSGAQEACA
RIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGVV
DPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGLDH
CSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPS
AIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSST
PEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALW
AWPSELSSPGPEASLPTEPAAQEKSLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPT
ETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRA
PEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVPASGDCVPSPCHNGGT
CLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYG
AHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLS
GENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVL
RYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKAL
LIPPSSPMPGP
(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.
NM.sub.--004442) Chan, J. and Watt, V. M., Oncogene 6 (6),
1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev.
Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000));
WO2003042661 (claim 12); WO200053216 (claim 1; Page 41);
WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (Page
128-132); WO200053216 (claim 1; Page 42);
Cross-references: MIM:600997; NP.sub.--004433.2;
NM.sub.--004442.sub.--1
TABLE-US-00025 [0795] 987 aa (SEQ ID NO: 22)
MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDENMNTIRTYQ
VCNVFESSQNNWLRTKFIRRRGAHRIHVEMKFSVRDCSSIPSVPGSCKETFNLYYYEADF
DSATKTFPNWMENPWVKVDTIAADESFSQVDLGGRVMKINTEVRSFGPVSRSGFYLAFQD
YGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLY
CNGDGEWLVPIGRCMCKAGFEAVENGTVCRGCPSGTFKANQGDEACTHCPINSRTTSEGA
TNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLMLEWTPPRDSGGREDLVYNI
ICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAVNGVTDQSPF
SPQFASVNITTNQAAPSAVSIMHQVSRTVDSITLSWSQPDQPNGVILDYELQYYEKELSE
YNATAIKSPTNTVTVQGLKAGAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIQEK
LPLIIGSSAAGLVFLIAVVVIAIVCNRRRGFERADSEYTDKLQHYTSGHMTPGMKIYIDP
FTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEVCSGHLKLPGKREIFVAIKTLKSGY
TEKQRRDFLSEASIMGQFDHPNVIHLEGVVTKSTPVMIITEFMENGSLDSFLRQNDGQFT
VIQLVGMLRGIAAGMKYLADMNYVHRDLAARNILVNSNLVCKVSDFGLSRFLEDDTSDPT
YTSALGGKIPIRWTAPEAIQYRKFTSASDVWSYGIVMWEVMSYGERPYWDMTNQDVINAI
EQDYRLPPPMDCPSALHQLMLDCWQKDRNHRPKFGQIVNTLDKMIRNPNSLKAMAPLSSG
INLPLLDRTIPDYTSFNIVDEWLEAIKMGQYKESFANAGFTSFDVVSQMMMEDILRVGVT
LAGHQKKILNSIQVMRAQMNQIQSVEV
(23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899
(claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3);
US2003165504 (claim 1); US2003124140 (Example 2); US2003065143
(FIG. 60); WO2002102235 (claim 13; Page 299); US2003091580 (Example
2); WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG.
7b); WO200202624 (claim 13; FIG. 1A-1B); US2002034749 (claim 54;
Page 45-46); WO200206317 (Example 2; Page 320-321, claim 34; Page
321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; FIG.
1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2;
Page 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1);
WO200271928 (Page 233-234, 452-453); WO 0116318;
TABLE-US-00026 282 aa (SEQ ID NO: 23)
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEP
DIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV
QLTDAGTYKCYIITSKGKKNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVV
WASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKV
TESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK
(24) PSCA (Prostate stem cell antigen precursor, Genbank accession
no. AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci. USA. 95,
1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000;
Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709;
EP1394274 (Example 11); US2004018553 (claim 17); WO2003008537
(claim 1); WO200281646 (claim 1; Page 164); WO2003003906 (claim 10;
Page 288); WO200140309 (Example 1; FIG. 17); US2001055751 (Example
1; FIG. 1b); WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17;
Page 97); WO9851824 (claim 10; Page 94); WO9840403 (claim 2; FIG.
1B);
Accession: 043653; EMBL; AF043498; AAC39607.1.
TABLE-US-00027 [0796] 123 aa (SEQ ID NO: 24)
MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLT
VISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGP
GQL
(25) GEDA (Genbank accession No. AY260763); AAP 14954 lipoma HMGIC
fusion-partner-like protein/pid=AAP14954.1-Homo sapiens Species:
Homo sapiens (human) WO2003054152 (claim 20); WO2003000842 (claim
1); WO2003023013 (Example 3, claim 20); US2003194704 (claim
45);
Cross-references: GI:30102449; AAP14954.1; AY260763.sub.--1
TABLE-US-00028 [0797] 236 aa (SEQ ID NO: 25)
MPGAAAAAAAAAAAMLPAQEAAKLYHTNYVRNSRAIGVLWAIFTICFAIVNVVCFIQPYW
IGDGVDTPQAGYFGLFHYCIGNGFSRELTCRGSFTDFSTLPSGAFKAASFFIGLSMMLII
ACIICFTLFFFCNTATVYKICAWMQLTSAACLVLGCMIFPDGWDSDEVKRMCGEKTDKYT
LGACSVRWAYILAIIGILDALILSFLAFVLGNRQDSLMAEELKAENKVLLSQYSLE
(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3,
BR3, Genbank accession No. NP.sub.--443177.1); NP.sub.--443177 BAFF
receptor/pid=NP.sub.--443177.1-Homo sapiens Thompson, J. S., et al.
Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611;
WO2003045422 (Example; Page 32-33); WO2003014294 (claim 35; FIG.
6B); WO2003035846 (claim 70; Page 615-616); WO200294852 (Col
136-137); WO200238766 (claim 3; Page 133); WO200224909 (Example 3;
FIG. 3);
Cross-references: MIM:606269; NP 443177.1;
NM.sub.--052945.sub.--1
TABLE-US-00029 [0798] 184 aa (SEQ ID NO: 26)
MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQ
ESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGD
KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAG
PEQQ
(27) CD22 (B-cell receptor CD22-B isoform, Genbank accession No.
NP-001762.1); Stamenkovic, I. and Seed, B., Nature 345 (6270),
74-77 (1990); US2003157113; US2003118592; WO2003062401 (claim 9);
WO2003072036 (claim 1; FIG. 1); WO200278524 (Example 2);
Cross-references: MIM:107266; NP.sub.--001762.1;
NM.sub.--001771.sub.--1
TABLE-US-00030 [0799] 847 aa (SEQ ID NO: 27)
MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFH
NPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLR
MESKTEKWMERIHLNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEG
VPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKH
TPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT
KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPL
PTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPK
KVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNT
TIACARCNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQ
FFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM
SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPHHSQKLRLEPVKVQHSGAYWCQ
GTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQG
LQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLREPEMNIPRTGDAES
SEMQRPPRTCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENV
DYVILKH
(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation) PROTEIN
SEQUENCE Full mpggpgv . . . dvqlekp (SEQ ID NO:28) (1.226; 226 aa),
pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank
accession No. NP.sub.--001774.1; WO2003088808, US20030228319;
WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14);
WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No.
5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller
et al. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al.
(1994) Immunogenetics 40(4):287-295; Preud'homme et al. (1992)
Clin. Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol.
148(2) 633-637; Sakaguchi et al. (1988) EMBO J.
7(11):3457-3464;
TABLE-US-00031 226 aa (SEQ ID NO: 28)
MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSN
NANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCG
TYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGD
EYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled
receptor that is activated by the CXCL13 chemokine, functions in
lymphocyte migration and humoral defense, plays a role in HIV-2
infection and perhaps development of AIDS, lymphoma, myeloma, and
leukemia) PROTEIN SEQUENCE Full mnyphl . . . atshtf (1.372; 372
aa), pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank
accession No. NP.sub.--001707.1; WO2004040000; WO2004015426;
US2003105292 (Example 2); U.S. Pat. No. 6,555,339 (Example 2);
WO200261087 (FIG. 1); WO200157188 (claim 20, page 269); WO200172830
(pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2,
pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat. No.
5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);
WO9217497 (claim 7, FIG. 5); Dobner et al. (1992) Eur. J. Immunol.
22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779;
TABLE-US-00032 372 aa (SEQ ID NO: 29)
MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSL
IFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTF
LCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLL
ALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVG
VVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSL
PVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSL
SESENATSLTTF
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)
that binds peptides and presents them to CD4+ T lymphocytes)
PROTEIN SEQUENCE Full mgsgwvp . . . vllpqsc (SEQ ID NO:30) (1.273;
273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3,
Genbank accession No. NP.sub.--002111.1; Tonnelle et al. (1985)
EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics
29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441;
Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903;
Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al.
(1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) Tissue Antigens
59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No. 6,153,408
(Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat. No.
6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics
30(1):66-68; Larhammar et al. (1985) J. Biol. Chem.
260(26):14111-14119;
TABLE-US-00033 273 aa (SEQ ID NO: 30)
MGSGWVPWVVALLVNLTRLDSSMTQGTDSPEDFVIQAKADCYFTNGTEKVQFVVRFIFNL
EEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQAVDGVCRHNYRLGAPFTVGRK
VQPEVTVYPERTPLLHQHNLLHCSVTGFYPGDIKIKWFLNGQEERAGVMSTGPIRNGDWT
FQTVVMLEMTPELGHVYTCLVDHSSLLSPVSVEWRAQSEYSWRKMLSGIAAELLGLIFLL
VGIVIQLRAQKGYVRTQMSGNEVSRAVLLPQSC
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an
ion channel gated by extracellular ATP, may be involved in synaptic
transmission and neurogenesis, deficiency may contribute to the
pathophysiology of idiopathic detrusor instability) PROTEIN
SEQUENCE Full mgqagck . . . lephrst (SEQ ID NO:31) (1.422; 422 aa),
pI: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank
accession No. NP.sub.--002552.2; Le et al. (1997) FEBS Lett.
418(1-2):195-199; WO2004047749; WO2003072035 (claim 10); Touchman
et al. (2000) Genome Res. 10:165-173; WO200222660 (claim 20);
WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page
82);
TABLE-US-00034 422 aa (SEQ ID NO: 31)
MGQAGCKGLCLSLFDYKTEKYVIAKNKKVGLLYRLLQASILAYLVVWVFLIKKGYQDVDT
SLQSAVITKVKGVAFTNTSDLGQRIWDVADYVIPAQGENVFFVVTNLIVTPNQRQNVCAE
NEGIPDGACSKDSDCHAGEAVTAGNGVKTGRCLRRENLARGTCEIFAWCPLETSSRPEEP
FLKEAEDFTIFIKNHIRFPKFNFSKSNVMDVKDRSFLKSCHFGPKNHYCPIFRLGSVIRW
AGSDFQDIALEGGVIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSVSSGYNFRFARY
YRDAAGVEFRTLMKAYGIRFDVMVNGKGAFFCDLVLIYLIKKREFYRDKKYEEVRGLEDS
SQEAEDEASGLGLSEQLTSGPGLLGMPEQQELQEPPEAKRGSSSQKGNGSVCPQLLEPHR ST
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN
SEQUENCE Full maeaity . . . tafrfpd (SEQ ID NO:32) (1.359; 359 aa),
pI: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank
accession No. NP.sub.--001773.1; WO2004042346 (claim 65);
WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von
Hoegen et al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et
al. (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903;
TABLE-US-00035 359 aa (SEQ ID NO: 32)
MAEAITYADLRFVKAPLKKSISSRLGQDPGADDDGEITYENVQVPAVLGVPSSLASSVLG
DKAAVKSEQPTASWRAVTSPAVGRILPCRTTCLRYLLLGLLLTCLLLGVTAICLGVRYLQ
VSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQA
AEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMH
QKSCFYISLTSKNWQESQKQCETLSSKLATFSEIYPQSHSYYFLNSLLPNGGSGNSYWTG
LSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICEMTAFRFPD
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis) PROTEIN SEQUENCE Full mafdvsc . . . rwkyqhi (SEQ ID
NO:33) (1.661; 661 aa), pI: 6.20, MW: 74147 TM: 1 [P] Gene
Chromosome: 5q12, Genbank accession No. NP.sub.--005573.1;
US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al.,
(1996) Genomics 38(3):299-304; Miura et al. (1998) Blood
92:2815-2822; WO2003083047; WO9744452 (pages 24-26);
TABLE-US-00036 661 aa (SEQ ID NO: 33)
MAFDVSCFFWVVLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDTLPNTTEFLEF
SFNFLPTIHNRTFSRLMNLTFLDLTRCQINWIHEDTFQSHHQLSTLVLTGNPLIFMAETS
LNGPKSLKHLFLIQTGISNLEFIPVHNLENLESLYLGSNHISSIKFPKDFPARNLKVLDF
QNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIELGAFDSTVFQSLNFGGTPNLSVIFN
GLQNSTTQSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEHRFSDISSTTFQCFTQL
QELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDQLCOISAANFPSLTHLYIRGNVKKLH
LGVGCLEKLGNLQTLDLSHNDIEASDCCSLQLKNLSHLQTLNLSHNEPLGLQSQAFKECP
QLELLDLAFTRLHINAPQSPFQNLHFLQVLNLTYCFLDTSNQHLLAGLPVLRHLNLKGNH
FQDGTITKTNLLQTVGSLEVLILSSCGLLSIDQQAFHSLGKMSHVDLSHNSLTCDSIDSL
SHLKGIYLNLAANSINIISPRLLPILSQQSTINLSHNPLDCTCSNIHFLTWYKENLHKLE
GSEETTCANPPSLRGVKLSDVKLSCGITAIGIFFLIVFLLLLAILLFFAVKYLLRWKYQH I
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the
immunoglobulin Fc domain that contains C2 type Ig-like and ITAM
domains, may have a role in B-lymphocyte differentiation) PROTEIN
SEQUENCE Full m1pr111 . . . vdyedam (SEQ ID NO:34) (1.429; 429 aa),
p1: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank
accession No. NP.sub.--443170.1; WO2003077836; WO200138490 (claim
6, FIG. 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci.
USA 98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1);
WO2003089624 (claim 7);
TABLE-US-00037 429 aa (SEQ ID NO: 34)
MLPRLLLLICAPLCEPAELFLIASPSHPTEGSPVTLTCKMPFLQSSDAQFQFCFFRDTRA
LGPGWSSSPKLQIAAMWKEDTGSYWCEAQTMASKVLRSRRSQINVHRVPVADVSLETQPP
GGQVMEGDRLVLICSVAMGTGDITFLWYKGAVGLNLQSKTQRSLTAEYEIPSVRESDAEQ
YYCVAENGYGPSPSGLVSITVRIPVSRPILMLRAPRAQAAVEDVLELHCEALRGSPPILY
WFYHEDITLGSRSAPSGGGASFNLSLTEEHSGNYSCEANNGLGAQRSEAVTLNFTVPTGA
RSNHLTSGVIEGLLSTLGPATVALLECYGLKRKIGRRSARDPLRSLPSPLPQEFTYLNSP
TPGQLQPIYENVNVVSGDEVYSLAYYNQPEQESVAAETLGTHMEDKVSLDIYSRLRKANI
TDVDYEDAM
(35) IRTA2 (Immunoglobulin superfamily receptor translocation
associated 2, a putative immunoreceptor with possible roles in B
cell development and lymphomagenesis; deregulation of the gene by
translocation occurs in some B cell malignancies) PROTEIN SEQUENCE
Full mllwvil . . . assaphr (SEQ ID NO:35) (1.977; 977 aa), pI: 6.88
MW: 106468 TM: 1 [P] Gene Chromosome: 1q21, Genbank accession No.
NP.sub.--112571.1; WO2003024392 (claim 2, FIG. 97); Nakayama et al.
(2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836;
WO200138490 (claim 3, FIG. 18B-1-18B-2);
TABLE-US-00038 977 aa (SEQ ID NO: 35)
MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRFYSPQKTKWYHR
YLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDFSSASLILQAPLSVFEGDSVV
LRCRAKAEVTLNNTIYKNDNVLAFLNKRTDFHIPHACLKDNGAYRCTGYKESCCPVSSNT
VKIQVQEPFTRPVLRASSFQPISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWS
LSPNFQITAMWSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTLSPEKALNFE
GTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNYYCTADNG
LGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLHCEAQRGSLPILYQFHHEDAA
LERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSITVPVSHPVLTLSSA
EALTFEGATVTLHCEVQRGSPQILYQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNYY
CTADNGFGPQRSEVVSLFVTVPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWF
YHEDVTLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAQHSDTISLSVIVPVSRPI
LTFRAPRAQAVVGDLLELHCEALRGSSPILYWFYHEDVTLGKISAPSGGGASFNLSLTTE
HSGIYSCEADNGPEAQRSEMVTLKVAVPVSRPVLTLRAPGTHAAVGDLLELHCEALRGSP
LILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGL
TANRSGPFATGVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSPPDSDSQEPTYH
NVPAWEELQPVYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPITYSEVKVA
STPVSGSLFLASSAPHR
[0800] See also: WO04/045516 (3 Jun. 2004); WO03/000113 (3 Jan.
2003); WO02/016429 (28 Feb. 2002); WO02/16581 (28 Feb. 2002);
WO03/024392 (27 Mar. 2003); WO04/016225 (26 Feb. 2004); WO01/40309
(7 Jun. 2001), and U.S. Provisional patent application Ser. No.
60/520,842 "COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF
HEMATOPOIETIC ORIGIN", filed 17 Nov. 2003; all of which are
incorporated herein by reference in their entirety.
[0801] In an embodiment, the Ligand-Linker-Drug Conjugate has
Formula IIIa, where the Ligand is an antibody Ab including one that
binds at least one of CD30, CD40, CD70, Lewis Y antigen, w=0, y=0,
and D has Formula Ib. Exemplary Conjugates of Formula IIIa include
where R.sup.17 is --(CH.sub.2).sub.5--. Also included are such
Conjugates of Formula IIIa in which D has the structure of Compound
2 in Example 3 and esters thereof. Also included are such
Conjugates of Formula IIIa containing about 3 to about 8, in one
aspect, about 3 to about 5 Drug moieties D, that is, Conjugates of
Formula Ia wherein p is a value in the range about 3-8, for example
about 3-5. Conjugates containing combinations of the structural
features noted in this paragraph are also contemplated as within
the scope of the compounds of the invention.
[0802] In another embodiment, the Ligand-Linker-Drug Conjugate has
Formula IIIa, where Ligand is an Antibody Ab that binds one of
CD30, CD40, CD70, Lewis Y antigen, w=1, y=0, and D has Formula Ib.
Included are such Conjugates of Formula Ma in which R.sup.17 is
--(CH.sub.2).sub.5--. Also included are such Conjugates of Formula
IIIa in which W is -Val-Cit-, and/or where D has the structure of
Compound 2 in Example 3 and esters thereof. Also included are such
Conjugates of Formula IIIa containing about 3 to about 8,
preferably about 3 to about 5 Drug moieties D, that is, Conjugates
of Formula Ia wherein p is a value in the range of about 3-8,
preferably about 3-5. Conjugates containing combinations of the
structural features noted in this paragraph are also exemplary.
[0803] In an embodiment, the Ligand-Linker-Drug Conjugate has
Formula IIIa, where the Ligand is an Antibody Ab that binds one of
CD30, CD40, CD70, Lewis Y antigen, w=1, y=1, and D has Formula Ib.
Included are Conjugates of Formula IIIa in which R.sup.17 is
--(CH.sub.2).sub.5--. Also included are such Conjugates of Formula
IIIa where: W is -Val-Cit-; Y has Formula X; D has the structure of
Compound 2 in Example 3 and esters thereof; p is about 3 to about
8, preferably about 3 to about 5 Drug moieties D. Conjugates
containing combinations of the structural features noted in this
paragraph are also contemplated within the scope of the compounds
of the invention.
[0804] A further embodiment is an antibody drug conjugate (ADC), or
a pharmaceutically acceptable salt or solvate thereof, wherein Ab
is an antibody that binds one of the tumor-associated antigens
(1)-(35) noted above (the "TAA Compound").
[0805] Another embodiment is the TAA Compound or pharmaceutically
acceptable salt or solvate thereof that is in isolated and purified
form.
[0806] Another embodiment is a method for killing or inhibiting the
multiplication of a tumor cell or cancer cell comprising
administering to a patient, for example a human with a
hyperproliferative disorder, an amount of the TAA Compound or a
pharmaceutically acceptable salt or solvate thereof, said amount
being effective to kill or inhibit the multiplication of a tumor
cell or cancer cell.
[0807] Another embodiment is a method for treating cancer
comprising administering to a patient, for example a human with a
hyperproliferative disorder, an amount of the TAA Compound or a
pharmaceutically acceptable salt or solvate thereof, said amount
being effective to treat cancer, alone or together with an
effective amount of an additional anticancer agent.
[0808] Another embodiment is a method for treating an autoimmune
disease, comprising administering to a patient, for example a human
with a hyperproliferative disorder, an amount of the TAA Compound
or a pharmaceutically acceptable salt or solvate thereof, said
amount being effective to treat an autoimmune disease.
[0809] The antibodies suitable for use in the invention can be
produced by any method known in the art for the synthesis of
antibodies, in particular, by chemical synthesis or by recombinant
expression, and are preferably produced by recombinant expression
techniques.
4.5.1 Production of Recombinant Antibodies
[0810] Antibodies of the invention can be produced using any method
known in the art to be useful for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression.
[0811] Recombinant expression of antibodies, or fragment,
derivative or analog thereof, requires construction of a nucleic
acid that encodes the antibody. If the nucleotide sequence of the
antibody is known, a nucleic acid encoding the antibody may be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligation of those oligonucleotides, and then amplification of the
ligated oligonucleotides, e.g., by PCR.
[0812] Alternatively, a nucleic acid molecule encoding an antibody
can be generated from a suitable source. If a clone containing the
nucleic acid encoding the particular antibody is not available, but
the sequence of the antibody is known, a nucleic acid encoding the
antibody can be obtained from a suitable source (e.g., an antibody
cDNA library, or cDNA library generated from any tissue or cells
expressing the immunoglobulin) by, e.g., PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence.
[0813] If an antibody that specifically recognizes a particular
antigen is not commercially available (or a source for a cDNA
library for cloning a nucleic acid encoding such an
immunoglobulin), antibodies specific for a particular antigen can
be generated by any method known in the art, for example, by
immunizing a patient, or suitable animal model such as a rabbit or
mouse, to generate polyclonal antibodies or, more preferably, by
generating monoclonal antibodies, e.g., as described by Kohler and
Milstein (1975, Nature 256:495-497) or, as described by Kozbor et
al. (1983, Immunology Today 4:72) or Cole et al. (1985 in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Alternatively, a clone encoding at least the Fab portion of
the antibody can be obtained by screening Fab expression libraries
(e.g., as described in Huse et al., 1989, Science 246:1275-1281)
for clones of Fab fragments that bind the specific antigen or by
screening antibody libraries (See, e.g., Clackson et al., 1991,
Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA
94:4937).
[0814] Once a nucleic acid sequence encoding at least the variable
domain of the antibody is obtained, it can be introduced into a
vector containing the nucleotide sequence encoding the constant
regions of the antibody (see, e.g., International Publication No.
WO 86/05807; WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors
containing the complete light or heavy chain that allow for the
expression of a complete antibody molecule are available. Then, the
nucleic acid encoding the antibody can be used to introduce the
nucleotide substitutions or deletion necessary to substitute (or
delete) the one or more variable region cysteine residues
participating in an intrachain disulfide bond with an amino acid
residue that does not contain a sulfhydryl group. Such
modifications can be carried out by any method known in the art for
the introduction of specific mutations or deletions in a nucleotide
sequence, for example, but not limited to, chemical mutagenesis and
in vitro site directed mutagenesis (Hutchinson et al., 1978, J.
Biol. Chem. 253:6551).
[0815] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. A chimeric antibody is a molecule in which
different portions are derived from different animal species, such
as those having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0816] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988,
Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be
adapted to produce single chain antibodies. Single chain antibodies
are formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional Fv fragments
in E. coli may also be used (Skerra et al., 1988, Science
242:1038-1041).
[0817] Antibody fragments that recognize specific epitopes can be
generated by known techniques. For example, such fragments include,
but are not limited to the F(ab').sub.2 fragments that can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments that can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments.
[0818] Once a nucleic acid sequence encoding an antibody has been
obtained, the vector for the production of the antibody can be
produced by recombinant DNA technology using techniques well known
in the art. Methods that are well known to those skilled in the art
can be used to construct expression vectors containing the antibody
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. See, for example, the techniques described
in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual,
2.sup.nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in
Molecular Biology, John Wiley & Sons, NY).
[0819] An expression vector comprising the nucleotide sequence of
an antibody or the nucleotide sequence of an antibody can be
transferred to a host cell by conventional techniques (e.g.,
electroporation, liposomal transfection, and calcium phosphate
precipitation), and the transfected cells are then cultured by
conventional techniques to produce the antibody. In specific
embodiments, the expression of the antibody is regulated by a
constitutive, an inducible or a tissue, specific promoter.
[0820] The host cells used to express the recombinant antibody can
be either bacterial cells such as Escherichia coli, or, preferably,
eukaryotic cells, especially for the expression of whole
recombinant immunoglobulin molecule. In particular, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al.,
1990, BioTechnology 8:2).
[0821] A variety of host-expression vector systems can be utilized
to express the immunoglobulin antibodies. Such host-expression
systems represent vehicles by which the coding sequences of the
antibody can be produced and subsequently purified, but also
represent cells that can, when transformed or transfected with the
appropriate nucleotide coding sequences, express an antibody
immunoglobulin molecule in situ. These include, but are not limited
to, microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing immunoglobulin coding
sequences; yeast (e.g., Saccharomyces Pichia) transformed with
recombinant yeast expression vectors containing immunoglobulin
coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the
immunoglobulin coding sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing immunoglobulin coding sequences; or mammalian cell
systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
[0822] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
antibody being expressed. For example, when a large quantity of
such a protein is to be produced, vectors that direct the
expression of high levels of fusion protein products that are
readily purified might be desirable. Such vectors include, but are
not limited, to the E. coli expression vector pUR278 (Ruther et
al., 1983, EMBO J. 2:1791), in which the antibody coding sequence
may be ligated individually into the vector in frame with the lac Z
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the
like. pGEX Vectors can also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption and binding to a matrix
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0823] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) or the analogous virus from Drosophila
Melanogaster is used as a vector to express foreign genes. The
virus grows in Spodoptera frugiperda cells. The antibody coding
sequence can be cloned individually into non-essential regions (for
example the polyhedrin gene) of the virus and placed under control
of an AcNPV promoter (for example the polyhedrin promoter).
[0824] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest can be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) results in a
recombinant virus that is viable and capable of expressing the
immunoglobulin molecule in infected hosts. (e.g., see Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals can also be required for efficient translation
of inserted antibody coding sequences. These signals include the
ATG initiation codon and adjacent sequences. Furthermore, the
initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression can be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:51-544).
[0825] In addition, a host cell strain can be chosen to modulate
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells that possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product can be used. Such mammalian
host cells include, but are not limited to, CHO, VERY, BH, Hela,
COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and
T47D, CRL7030 and Hs578Bst.
[0826] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express an antibody can be engineered. Rather than
using expression vectors that contain viral origins of replication,
host cells can be transformed with DNA controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of the foreign DNA,
engineered cells can be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci that in turn can be cloned
and expanded into cell lines. This method can advantageously be
used to engineer cell lines which express the antibody. Such
engineered cell lines can be particularly useful in screening and
evaluation of tumor antigens that interact directly or indirectly
with the antibody.
[0827] A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
DHFR, which confers resistance to methotrexate (Wigler et al.,
1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIB TECH 11(5):155-215) and hygro, which confers resistance
to hygromycin (Santerre et al., 1984, Gene 30:147). Methods
commonly known in the art of recombinant DNA technology which can
be used are described in Ausubel et al. (eds., 1993, Current
Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.
(eds), 1994, Current Protocols in Human Genetics, John Wiley &
Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).
[0828] The expression levels of an antibody can be increased by
vector amplification (for a review, see Bebbington and Hentschel,
The use of vectors based on gene amplification for the expression
of cloned genes in mammalian cells in DNA cloning, Vol. 3.
(Academic Press, New York, 1987)). When a marker in the vector
system expressing an antibody is amplifiable, an increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the nucleotide sequence of the antibody,
production of the antibody will also increase (Crouse et al., 1983,
Mol. Cell. Biol. 3:257).
[0829] The host cell can be co-transfected with two expression
vectors, the first vector encoding a heavy chain derived
polypeptide and the second vector encoding a light chain derived
polypeptide. The two vectors can contain identical selectable
markers that enable equal expression of heavy and light chain
polypeptides. Alternatively, a single vector can be used to encode
both heavy and light chain polypeptides. In such situations, the
light chain should be placed before the heavy chain to avoid an
excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52;
Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding
sequences for the heavy and light chains can comprise cDNA or
genomic DNA.
[0830] Once the antibody has been recombinantly expressed, it can
be purified using any method known in the art for purification of
an antibody, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after
Protein A, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins.
[0831] In yet another exemplary embodiment, the antibody is a
monoclonal antibody.
[0832] In any case, the hybrid antibodies have a dual specificity,
preferably with one or more binding sites specific for the hapten
of choice or one or more binding sites specific for a target
antigen, for example, an antigen associated with a tumor, an
autoimmune disease, an infectious organism, or other disease
state.
4.5.2 Production of Antibodies
[0833] The production of antibodies will be illustrated with
reference to anti-CD30 antibodies but it will be apparent for those
skilled in the art that antibodies to other members of the TNF
receptor family can be produced and modified in a similar manner.
The use of CD30 for the production of antibodies is exemplary only
and not intended to be limiting.
[0834] The CD30 antigen to be used for production of antibodies may
be, e.g., a soluble form of the extracellular domain of CD30 or a
portion thereof, containing the desired epitope. Alternatively,
cells expressing CD30 at their cell surface (e.g., L540 (Hodgkin's
lymphoma derived cell line with a T cell phenotype) and L428
(Hodgkin's lymphoma derived cell line with a B cell phenotype)) can
be used to generate antibodies. Other forms of CD30 useful for
generating antibodies will be apparent to those skilled in the
art.
[0835] In another exemplary embodiment, the ErbB2 antigen to be
used for production of antibodies may be, e.g., a soluble form of
the extracellular domain of ErbB2 or a portion thereof, containing
the desired epitope. Alternatively, cells expressing ErbB2 at their
cell surface (e.g., NIH-3T3 cells transformed to overexpress ErbB2;
or a carcinoma cell line such as SK-BR-3 cells, see Stancovski et
al. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991)) can be used to
generate antibodies. Other forms of ErbB2 useful for generating
antibodies will be apparent to those skilled in the art.
(i) Polyclonal Antibodies
[0836] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C=NR, where R and
R.sup.1 are different alkyl groups.
[0837] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
(ii) Monoclonal Antibodies
[0838] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0839] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0840] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0841] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0842] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987)).
[0843] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0844] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0845] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0846] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188
(1992).
[0847] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0848] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy chain and light chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; and Morrison, et al. (1984) Proc. Natl. Acad. Sci. USA
81:6851), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0849] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
(iii) Humanized Antibodies
[0850] A humanized antibody may have one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science 239:1534-1536 (1988)), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567) wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0851] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0852] In another embodiment, the antibodies may be humanized with
retention of high affinity for the antigen and other favorable
biological properties. Humanized antibodies may be prepared by a
process of analysis of the parental sequences and various
conceptual humanized products using three-dimensional models of the
parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in
the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0853] Various forms of the humanized antibody are contemplated.
For example, the humanized antibody may be an antibody fragment,
such as a Fab. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0854] The Examples describe production of an exemplary humanized
anti-ErbB2 antibody. The humanized antibody may, for example,
comprise nonhuman hypervariable region residues incorporated into a
human variable heavy domain and may further comprise a framework
region (FR) substitution at a position selected from the group
consisting of 69H, 71H and 73H utilizing the variable domain
numbering system set forth in Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). In one embodiment, the
humanized antibody comprises FR substitutions at two or all of
positions 69H, 71H and 73H. Another Example describes preparation
of purified trastuzumab antibody from the HERCEPTIN.RTM.
formulation.
(iv) Human Antibodies
[0855] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and
5,545,807.
[0856] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905. As discussed above, human antibodies may also be
generated by in vitro activated B cells (see U.S. Pat. Nos.
5,567,610 and 5,229,275). Human anti-CD30 antibodies are described
in U.S. patent application Ser. No. 10/338,366.
(v) Antibody Fragments
[0857] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example. Such
linear antibody fragments may be monospecific or bispecific.
(vi) Bispecific Antibodies
[0858] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
CD30 protein. Alternatively, an anti-CD30 arm may be combined with
an arm which binds to a Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so
as to focus cellular defense mechanisms to the CD30-expressing
cell. Bispecific antibodies may also be used to localize cytotoxic
agents to cells which express CD30.
[0859] Traditional production of full length bispecific antibodies
is based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, where the two chains have different
specificities (Millstein et al., Nature, 305:537-539 (1983)).
Because of the random assortment of immunoglobulin heavy and light
chains, these hybridomas (quadromas) produce a potential mixture of
10 different antibody molecules, of which only one has the correct
bispecific structure. Purification of the correct molecule, which
is usually done by affinity chromatography steps, is rather
cumbersome, and the product yields are low. Similar procedures are
disclosed in WO 93/08829, and in Traunecker et al., EMBO J.,
10:3655-3659 (1991). According to a different approach, antibody
variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. It is preferred to have
the first heavy-chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0860] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0861] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the CH3 domain of an antibody constant
domain. In this method, one or more small amino acid side chains
from the interface of the first antibody molecule are replaced with
larger side chains (e.g., tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s)
are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g.,
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
[0862] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0863] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody.
[0864] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0865] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
(vii) Other Amino Acid Sequence Modifications
[0866] Amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. Amino acid sequence variants of the antibodies are
prepared by introducing appropriate nucleotide changes into the
antibody nucleic acid, or by peptide synthesis. Such modifications
include, for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites.
[0867] A useful method for identification of certain residues or
regions of the antibody that are favored locations for mutagenesis
is called "alanine scanning mutagenesis" as described by Cunningham
and Wells Science, 244:1081-1085 (1989). Here, a residue or group
of target residues are identified (e.g., charged residues such as
arg, asp, his, lys, and glu) and replaced by a neutral or
negatively charged amino acid (most preferably alanine or
polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
[0868] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g., for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0869] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also
contemplated.
[0870] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally-occurring residues are
divided into groups based on common side-chain properties:
[0871] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0872] (2) neutral hydrophilic: cys, ser, thr;
[0873] (3) acidic: asp, glu;
[0874] (4) basic: asn, gln, his, lys, arg;
[0875] (5) residues that influence chain orientation: gly, pro;
and
[0876] (6) aromatic: trp, tyr, phe.
[0877] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0878] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g., a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and the antigen. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0879] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al. J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J.
Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug
Design 3:219-230 (1989).
[0880] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
(viii) Glycosylation Variants
[0881] Antibodies in the ADC of the invention may be glycosylated
at conserved positions in their constant regions (Jefferis and
Lund, (1997) Chem. Immunol. 65:111-128; Wright and Morrison, (1997)
TibTECH 15:26-32). The oligosaccharide side chains of the
immunoglobulins affect the protein's function (Boyd et al., (1996)
Mol. Immunol. 32:1311-1318; Wittwe and Howard, (1990) Biochem.
29:4175-4180), and the intramolecular interaction between portions
of the glycoprotein which can affect the conformation and presented
three-dimensional surface of the glycoprotein (Hefferis and Lund,
supra; Wyss and Wagner, (1996) Current Opin. Biotech. 7:409-416).
Oligosaccharides may also serve to target a given glycoprotein to
certain molecules based upon specific recognition structures. For
example, it has been reported that in agalactosylated IgG, the
oligosaccharide moiety `flips` out of the inter-CH2 space and
terminal N-acetylglucosamine residues become available to bind
mannose binding protein (Malhotra et al., (1995) Nature Med.
1:237-243). Removal by glycopeptidase of the oligosaccharides from
CAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibody
which recognizes the CDw52 antigen of human lymphocytes) produced
in Chinese Hamster Ovary (CHO) cells resulted in a complete
reduction in complement mediated lysis (CMCL) (Boyd et al., (1996)
Mol. Immunol. 32:1311-1318), while selective removal of sialic acid
residues using neuraminidase resulted in no loss of DMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression off
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al. (1999) Mature
Biotech. 17:176-180).
[0882] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0883] Glycosylation variants of antibodies are variants in which
the glycosylation pattern of an antibody is altered. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, adding one or more carbohydrate moieties to the antibody,
changing the composition of glycosylation (glycosylation pattern),
the extent of glycosylation, etc.
[0884] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites). Similarly, removal of
glycosylation sites can be accomplished by amino acid alteration
within the native glycosylation sites of the antibody.
[0885] The amino acid sequence is usually altered by altering the
underlying nucleic acid sequence. These methods include, but are
not limited to, isolation from a natural source (in the case of
naturally-occurring amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the antibody.
[0886] The glycosylation (including glycosylation pattern) of
antibodies may also be altered without altering the amino acid
sequence or the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g., antibodies, as potential therapeutics is
rarely the native cell, significant variations in the glycosylation
pattern of the antibodies can be expected. See, e.g., Hse et al.,
(1997) J. Biol. Chem. 272:9062-9070. In addition to the choice of
host cells, factors which affect glycosylation during recombinant
production of antibodies include growth mode, media formulation,
culture density, oxygenation, pH, purification schemes and the
like. Various methods have been proposed to alter the glycosylation
pattern achieved in a particular host organism including
introducing or overexpressing certain enzymes involved in
oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261;
5,278,299). Glycosylation, or certain types of glycosylation, can
be enzymatically removed from the glycoprotein, for example using
endoglycosidase H (Endo H). In addition, the recombinant host cell
can be genetically engineered, e.g., make defective in processing
certain types of polysaccharides. These and similar techniques are
well known in the art.
[0887] The glycosylation structure of antibodies can be readily
analyzed by conventional techniques of carbohydrate analysis,
including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides.
4.5.2a Screening for Antibody-Drug Conjugates (ADC)
[0888] Transgenic animals and cell lines are particularly useful in
screening antibody drug conjugates (ADC) that have potential as
prophylactic or therapeutic treatments of diseases or disorders
involving overexpression of proteins including Lewis Y, CD30, CD40,
and CD70. Transgenic animals and cell lines are particularly useful
in screening antibody drug conjugates (ADC) that have potential as
prophylactic or therapeutic treatments of diseases or disorders
involving overexpression of HER2 (U.S. Pat. No. 6,632,979).
Screening for a useful ADC may involve administering candidate ADC
over a range of doses to the transgenic animal, and assaying at
various time points for the effect(s) of the ADC on the disease or
disorder being evaluated. Alternatively, or additionally, the drug
can be administered prior to or simultaneously with exposure to an
inducer of the disease, if applicable. Candidate ADC may be
screened serially and individually, or in parallel under medium or
high-throughput screening format. The rate at which ADC may be
screened for utility for prophylactic or therapeutic treatments of
diseases or disorders is limited only by the rate of synthesis or
screening methodology, including detecting/measuring/analysis of
data.
[0889] One embodiment is a screening method comprising (a)
transplanting cells from a stable renal cell cancer cell line into
a non-human animal, (b) administering an ADC drug candidate to the
non-human animal and (c) determining the ability of the candidate
to inhibit the formation of tumors from the transplanted cell
line.
[0890] Another embodiment is a screening method comprising (a)
contacting cells from a stable Hodgkin's disease cell line with an
ADC drug candidate and (b) evaluating the ability of the ADC
candidate to block ligand activation of CD40.
[0891] Another embodiment is a screening method comprising (a)
contacting cells from a stable Hodgkin's disease cell line with an
ADC drug candidate and (b) evaluating the ability of the ADC
candidate to induce cell death. In one embodiment the ability of
the ADC candidate to induce apoptosis is evaluated.
[0892] One embodiment is a screening method comprising (a)
transplanting cells from a stable cancer cell line into a non-human
animal, (b) administering an ADC drug candidate to the non-human
animal and (c) determining the ability of the candidate to inhibit
the formation of tumors from the transplanted cell line. The
invention also concerns a method of screening ADC candidates for
the treatment of a disease or disorder characterized by the
overexpression of HER2 comprising (a) contacting cells from a
stable breast cancer cell line with a drug candidate and (b)
evaluating the ability of the ADC candidate to inhibit the growth
of the stable cell line.
[0893] Another embodiment is a screening method comprising (a)
contacting cells from a stable cancer cell line with an ADC drug
candidate and (b) evaluating the ability of the ADC candidate to
block ligand activation of HER2. In one embodiment the ability of
the ADC candidate to block heregulin binding is evaluated. In
another embodiment the ability of the ADC candidate to block
ligand-stimulated tyrosine phosphorylation is evaluated.
[0894] Another embodiment is a screening method comprising (a)
contacting cells from a stable cancer cell line with an ADC drug
candidate and (b) evaluating the ability of the ADC candidate to
induce cell death. In one embodiment the ability of the ADC
candidate to induce apoptosis is evaluated.
[0895] Another embodiment is a screening method comprising (a)
administering an ADC drug candidate to a transgenic non-human
mammal that overexpresses in its mammary gland cells a native human
HER2 protein or a fragment thereof, wherein such transgenic mammal
has stably integrated into its genome a nucleic acid sequence
encoding a native human HER2 protein or a fragment thereof having
the biological activity of native human HER2, operably linked to
transcriptional regulatory sequences directing its expression to
the mammary gland, and develops a mammary tumor not responding or
poorly responding to anti-HER2 antibody treatment, or to a
non-human mammal bearing a tumor transplanted from said transgenic
non-human mammal; and (b) evaluating the effect of the ADC
candidate on the target disease or disorder. Without limitations,
the disease or disorder may be a HER2-overexpressing cancer, such
as breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic and bladder cancer. The cancer
preferably is breast cancer which expressed HER2 in at least about
500,000 copies per cell, more preferably at least about 2,000,000
copies per cell. ADC drug candidates may, for example, be evaluated
for their ability to induce cell death and/or apoptosis, using
assay methods well known in the art and described hereinafter.
[0896] In one embodiment, candidate ADC are screened by being
administered to the transgenic animal over a range of doses, and
evaluating the animal's physiological response to the compounds
over time. Administration may be oral, or by suitable injection,
depending on the chemical nature of the compound being evaluated.
In some cases, it may be appropriate to administer the compound in
conjunction with co-factors that would enhance the efficacy of the
compound. If cell lines derived from the subject transgenic animals
are used to screen for compounds useful in treating various
disorders, the test compounds are added to the cell culture medium
at an appropriate time, and the cellular response to the compound
is evaluated over time using the appropriate biochemical and/or
histological assays. In some cases, it may be appropriate to apply
the compound of interest to the culture medium in conjunction with
co-factors that would enhance the efficacy of the compound.
[0897] Thus, provided herein are assays for identifying ADC which
specifically target and bind a target protein, the presence of
which is correlated with abnormal cellular function, and in the
pathogenesis of cellular proliferation and/or differentiation that
is causally related to the development of tumors.
[0898] To identify an ADC which blocks ligand activation of an ErbB
(e.g., ErbB2) receptor, the ability of the compound to block ErbB
ligand binding to cells expressing the ErbB (ErbB2) receptor (e.g.,
in conjugation with another ErbB receptor with which the ErbB
receptor of interest forms an ErbB hetero-oligomer) may be
determined. For example, cells isolated from the transgenic animal
overexpressing HER2 and transfected to express another ErbB
receptor (with which HER2 forms hetero-oligomer) may be incubated,
i.e. culturing, with the ADC and then exposed to labeled ErbB
ligand. The ability of the compound to block ligand binding to the
ErbB receptor in the ErbB hetero-oligomer may then be
evaluated.
[0899] For example, inhibition of heregulin (HRG) binding to breast
tumor cell lines, overexpressing HER2 and established from the
transgenic non-human mammals (e.g., mice) herein, by the candidate
ADC may be performed using monolayer cultures on ice in a
24-well-plate format. Anti-ErbB2 monoclonal antibodies may be added
to each well and incubated for 30 minutes. .sup.125I-labeled
rHRG.beta.1.sub.177-224 (25,000 cpm) may then be added, and the
incubation may be continued for 4 to 16 hours. Dose response curves
may be prepared and an IC.sub.50 value (cytotoxic activity) may be
calculated for the compound of interest.
[0900] Alternatively, or additionally, the ability of an ADC to
block ErbB ligand-stimulated tyrosine phosphorylation of an ErbB
receptor present in an ErbB hetero-oligomer may be assessed. For
example, cell lines established from the transgenic animals herein
may be incubated with a test ADC and then assayed for ErbB
ligand-dependent tyrosine phosphorylation activity using an
anti-phosphotyrosine monoclonal antibody (which is optionally
conjugated with a detectable label). The kinase receptor activation
assay described in U.S. Pat. No. 5,766,863 is also available for
determining ErbB receptor activation and blocking of that activity
by the compound.
[0901] In one embodiment, one may screen for ADC which inhibit HRG
stimulation of p180 tyrosine phosphorylation in MCF7 cells
essentially as described below. For example, a cell line
established from a HER2-transgenic animal may be plated in 24-well
plates and the compound may be added to each well and incubated for
30 minutes at room temperature; then rHRG.beta..sub.1177-244 may be
added to each well to a final concentration of 0.2 nM, and the
incubation may be continued for about 8 minutes. Media may be
aspirated from each well, and reactions may be stopped by the
addition of 100 .mu.l of SDS sample buffer (5% SDS, 25 mM DTT, and
25 mM Tris-HCl, pH 6.8). Each sample (25 .mu.l) may be
electrophoresed on a 4-12% gradient gel (Novex) and then
electrophoretically transferred to polyvinylidene difluoride
membrane. Antiphosphotyrosine (at 1 .mu.g/ml) immunoblots may be
developed, and the intensity of the predominant reactive band at
M.sub.r-180,000 may be quantified by reflectance densitometry. An
alternate method to evaluate inhibition of receptor phosphorylation
is the KIRA (kinase receptor activation) assay of Sadick et al.
(1998) Jour. of Pharm. and Biomed. Anal. Some of the well
established monoclonal antibodies against HER2 that are known to
inhibit HRG stimulation of p180 tyrosine phosphorylation can be
used as positive control in this assay. A dose-response curve for
inhibition of HRG stimulation of p180 tyrosine phosphorylation as
determined by reflectance densitometry may be prepared and an
IC.sub.50 for the compound of interest may be calculated.
[0902] One may also assess the growth inhibitory effects of a test
ADC on cell lines derived from a HER2-transgenic animal, e.g.,
essentially as described in Schaefer et al. (1997) Oncogene
15:1385-1394. According to this assay, the cells may be treated
with a test compound at various concentrations for 4 days and
stained with crystal violet or the redox dye Alamar Blue.
Incubation with the compound may show a growth inhibitory effect on
this cell line similar to that displayed by monoclonal antibody 2C4
on MDA-MB-175 cells (Schaefer et al., supra). In a further
embodiment, exogenous HRG will not significantly reverse this
inhibition.
[0903] To identify growth inhibitory compounds that specifically
target an antigen of interest, one may screen for compounds which
inhibit the growth of cancer cells overexpressing antigen of
interest derived from transgenic animals, the assay described in
U.S. Pat. No. 5,677,171 can be performed. According to this assay,
cancer cells overexpressing the antigen of interst are grown in a
1:1 mixture of F12 and DMEM medium supplemented with 10% fetal
bovine serum, glutamine and penicillin streptomycin. The cells are
plated at 20,000 cells in a 35 mm cell culture dish (2 mls/35 mm
dish) and the test compound is added at various concentrations.
After six days, the number of cells, compared to untreated cells is
counted using an electronic COULTER.TM. cell counter. Those
compounds which inhibit cell growth by about 20-100% or about
50-100% may be selected as growth inhibitory compounds.
[0904] To select for compounds which induce cell death, loss of
membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD
uptake may be assessed relative to control. The PI uptake assay
uses cells isolated from the tumor tissueof interest of a
transgenic animal. According to this assay, the cells are cultured
in Dulbecco's Modified Eagle Medium (D-MEM):Ham's F-12 (50:50)
supplemented with 10% heat-inactivated FBS (Hyclone) and 2 mM
L-glutamine. Thus, the assay is performed in the absence of
complement and immune effector cells. The cells are seeded at a
density of 3.times.10.sup.6 per dish in 100.times.20 mm dishes and
allowed to attach overnight. The medium is then removed and
replaced with fresh medium alone or medium containing various
concentrations of the compound. The cells are incubated for a 3-day
time period. Following each treatment, monolayers are washed with
PBS and detached by trypsinization. Cells are then centrifuged at
1200 rpm for 5 minutes at 4.degree. C., the pellet resuspended in 3
ml cold Ca.sup.2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl,
2.5 mM CaCl.sub.2) and aliquoted into 35 mm strainer-capped
12.times.75 mm tubes (1 ml per tube, 3 tubes per treatment group)
for removal of cell clumps. Tubes then receive PI (10 .mu.g/ml).
Samples may be analyzed using a FACSCAN.TM. flow cytometer and
FACSCONVERT.TM. CellQuest software (Becton Dickinson). Those
compounds which induce statistically significant levels of cell
death as determined by PI uptake may be selected as cell
death-inducing compounds.
[0905] In order to select for compounds which induce apoptosis, an
annexin binding assay using cells established from the tumor tissue
of interest of the transgenic animal is performed. The cells are
cultured and seeded in dishes as discussed in the preceding
paragraph. The medium is then removed and replaced with fresh
medium alone or medium containing 10 .mu.g/ml of the antibody drug
conjugate (ADC). Following a three-day incubation period,
monolayers are washed with PBS and detached by trypsinization.
Cells are then centrifuged, resuspended in Ca.sup.2+ binding buffer
and aliquoted into tubes as discussed above for the cell death
assay. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1
.mu.g/ml). Samples may be analyzed using a FACSCAN.TM. flow
cytometer and FACSCONVERT.TM. CellQuest software (Becton
Dickinson). Those compounds which induce statistically significant
levels of annexin binding relative to control are selected as
apoptosis-inducing compounds.
4.5.3 In Vitro Cell Proliferation Assays
[0906] Generally, the cytotoxic or cytostatic activity of an
antibody drug conjugate (ADC) is measured by: exposing mammalian
cells having receptor proteins to the antibody of the ADC in a cell
culture medium; culturing the cells for a period from about 6 hours
to about 5 days; and measuring cell viability. Cell-based in vitro
assays were used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of
the ADC of the invention.
[0907] The in vitro potency of antibody drug conjugates was
measured by a cell proliferation assay (Example 18, FIGS. 7-10).
The CellTiter-Glo.RTM. Luminescent Cell Viability Assay is a
commercially available (Promega Corp., Madison, Wis.), homogeneous
assay method based on the recombinant expression of Coleoptera
luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and 5,700,670).
This cell proliferation assay determines the number of viable cells
in culture based on quantitation of the ATP present, an indicator
of metabolically active cells (Crouch et al. (1993) J. Immunol.
Meth. 160:81-88, U.S. Pat. No. 6,602,677). The CellTiter-Glo.RTM.
Assay was conducted in 96 well format, making it amenable to
automated high-throughput screening (HTS) (Cree et al. (1995)
AntiCancer Drugs 6:398-404). The homogeneous assay procedure
involves adding the single reagent (CellTiter-Glo.RTM. Reagent)
directly to cells cultured in serum-supplemented medium. Cell
washing, removal of medium and multiple pipetting steps are not
required. The system detects as few as 15 cells/well in a 384-well
format in 10 minutes after adding reagent and mixing. The cells may
be treated continuously with ADC, or they may be treated and
separated from ADC. Generally, cells treated briefly, i.e. 3 hours,
showed the same potency effects as continuously treated cells.
[0908] The homogeneous "add-mix-measure" format results in cell
lysis and generation of a luminescent signal proportional to the
amount of ATP present. The amount of ATP is directly proportional
to the number of cells present in culture. The CellTiter-Glo.RTM.
Assay generates a "glow-type" luminescent signal, produced by the
luciferase reaction, which has a half-life generally greater than
five hours, depending on cell type and medium used (FIG. 24).
Viable cells are reflected in relative luminescence units (RLU).
The substrate, Beetle Luciferin, is oxidatively decarboxylated by
recombinant firefly luciferase with concomitant conversion of ATP
to AMP and generation of photons. The extended half-life eliminates
the need to use reagent injectors and provides flexibility for
continuous or batch mode processing of multiple plates. This cell
proliferation assay can be used with various multiwell formats,
e.g., 96 or 384 well format. Data can be recorded by luminometer or
CCD camera imaging device. The luminescence output is presented as
relative light units (RLU), measured over time.
##STR00039##
[0909] The anti-proliferative effects of antibody drug conjugates
were measured by the cell proliferation, in vitro cell killing
assay above against four different breast tumor cell lines (FIGS.
7-10). IC.sub.50 values were established for SK-BR-3 and BT-474
which are known to over express HER2 receptor protein. Table 2a
shows the potency (IC.sub.50) measurements of exemplary antibody
drug conjugates in the cell proliferation assay against SK-BR-3
cells. Table 2b shows the potency (IC.sub.50) measurements of
exemplary antibody drug conjugates in the cell proliferation assay
against BT-474 cells.
[0910] Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAF, 3.8
MMAF/Ab; Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab;
Trastuzumab-MC-MMAF, 4.1 MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAE, 4.1
MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; and
Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab did not inhibit the
proliferation of MCF-7 cells (FIG. 9).
[0911] Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1
MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab;
Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab;
Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab;
Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; and
Trastuzumab-MC-MMAF, 4.1 MMAF/Ab did not inhibit the proliferation
of MDA-MB-468 cells (FIG. 10).
[0912] MCF-7 and MDA-MB-468 cells do not overexpress HER2 receptor
protein. The anti-HER2 antibody drug conjugates of the invention
therefore show selectivity for inhibition of cells which express
HER2.
TABLE-US-00039 TABLE 2a SK-BR-3 cells Antibody Drug Conjugate
IC.sub.50 (.mu.g ADC/ml) H-MC-MMAF, 4.1 MMAF/Ab 0.008 H-MC-MMAF,
4.8 MMAF/Ab 0.002 H-MC-vc-PAB-MMAE, 0.007 H-MC-vc-PAB-MMAE 0.015
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.0035-0.01 H-MC-vc-PAB-MMAF, 4.4
MMAF/Ab 0.006-0.007 H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab 0.006
H-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab 0.0035 H-MC-MMAF, 4.1 MMAF/Ab
0.0035 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.010 H-MC-vc-PAB-MMAF, 3.8
MMAF/Ab 0.007 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.015 H-MC-vc-PAB-MMAF,
3.7 MMAF/Ab. 0.010 H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab 0.0025 H-MC-MMAE,
8.8 MMAE/Ab 0.018 H-MC-MMAE, 4.6 MMAE/Ab 0.05
H-MC-(L)val-(L)cit-PAB-MMAE, 8.7 MMAE/Ab 0.0003
H-MC-(D)val-(D)cit-PAB-MMAE, 8.2 MMAE/Ab 0.02
H-MC-(D)val-(L)cit-PAB-MMAE, 8.4 MMAE/Ab 0.0015
H-MC-(D)val-(L)cit-PAB-MMAE, 3.2 MMAE/Ab 0.003 H-Trastuzumab 0.083
H-vc-MMAE, linked via a lysine [lys] 0.002 H-phe-lys-MMAE, linked
via a lysine [lys] 0.0015 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.004
Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab 0.01 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab
0.01 4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab 0.02 Anti-TF
Fab-MC-vc-PAB-MMAE* -- H = trastuzumab linked via a cysteine
[cys]except where noted
TABLE-US-00040 TABLE 2b BT474 cells Antibody Drug Conjugate
IC.sub.50 (.mu.g ADC/ml) H-MC-MMAF, 4.1 MMAF/Ab 0.008 H-MC-MMAF,
4.8 MMAF/Ab 0.002 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.015
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.02-0.05 H-MC-vc-PAB-MMAF, 4.4
MMAF/Ab 0.01 H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab 0.01 H-MC-vc-PAB-MMAE,
3.3 MMAE/Ab 0.02 H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab. 0.02
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.015 H-MC-(N-Me)vc-PAB-MMAF, 3.9
MMAF/Ab 0.010 H-MC-MMAF, 4.1 MMAF/Ab 0.00015 H-MC-vc-PAB-MMAE, 7.5
MMAE/Ab 0.0025 H-MC-MMAE, 8.8 MMAE/Ab 0.04 H-MC- MMAE, 4.6 MMAE/Ab
0.07 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.008 Hg-MC-vc-PAB-MMAF,
4.1 MMAF/Ab 0.01 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab 0.015 4D5
Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab 0.04 Anti-TF Fab-MC-vc-PAB-MMAE* --
H = trastuzumab linked via a cysteine [cys] H = trastuzumab 7C2 =
anti-HER2 murine antibody which binds a different epitope than
trastuzumab. Fc8 = mutant that does not bind to FcRn Hg =
"Hingeless" full-length humanized 4D5, with heavy chain hinge
cysteines mutated to serines. Expressed in E. coli (therefore
non-glycosylated.) Anti-TF Fab = anti-tissue factor antibody
fragment *activity against MDA-MB-468 cells
[0913] In a surprising and unexpected discovery, the in vitro cell
proliferation activity results of the ADC in Tables 2a and 2b show
generally that ADC with a low average number of drug moieties per
antibody showed efficacy, e.g., IC.sub.50<0.1 .mu.g ADC/ml. The
results suggest that at least for trastuzumab ADC, the optimal
ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5.
4.5.4 In Vivo Plasma Clearance and Stability
[0914] Pharmacokinetic plasma clearance and stability of ADC were
investigated in rats and cynomolgus monkeys. Plasma concentration
was measured over time. Table 2c shows pharmacokinetic data of
antibody drug conjugates and other dosed samples in rats. Rats are
a non-specific model for ErbB receptor antibodies, since the rat is
not known to express HER2 receptor proteins.
TABLE-US-00041 TABLE 2c Pharmacokinetics in Rats AUCinf CL T 1/2
Sample day* mL/day/ Cmax Term. % dose mg/kg .mu.g/mL kg .mu.g/mL
days Conj. H-MC-vc-PAB-MMAE 78.6 26.3 39.5 5.80 40.6 (Total Ab
H-MC-vc-PAB-MMAE 31.1 64.4 33.2 3.00 (Conj.) H-MC-vc-PAB-MMAF 170
12.0 47.9 8.4 50.0 (Total Ab) H-MC-vc-PAB-MMAF 83.9 24.0 44.7 4.01
(Conj.) H-MC-MMAE (Total Ab) 279 18.9 79.6 7.65 33 H-MC-MMAE
(Conj.) 90.6 62.9 62.9 4.46 5 mg/kg H-MC-MMAF (Total Ab) 299 6.74
49.1 11.6 37 H-MC-MMAF (Conj.) 110 18.26 50.2 4.54 H-MC-vc-MMAF,
306 6.6 78.7 11.9 19.6 wo/PAB, (Total Ab) H-MC-vc-MMAF, 59.9 33.4
82.8 2.1 wo/PAB, (Conj.) H-Me-vc-PAB-MMAF 186 10.8 46.9 8.3 45.3
(Total Ab) H-Me-vc-PAB-MMAF 84.0 23.8 49.6 4.3 (Conj.)
H-Me-vc-PAB-MMAE 135 15.0 44.9 11.2 23.8 (Total Ab)
H-Me-vc-PAB-MMAE 31.9 63.8 45.2 3.0 (Conj.) H-MC-vc-MMAF, 306 6.6
78.7 11.9 19.6 wo/PAB, (Total Ab) H-MC-vc-MMAF, 59.9 33.4 82.8 2.1
wo/PAB, (Conj.) H-MC-(D)val-(L)cit- 107 19.2 30.6 9.6 38.1 PAB-MMAE
(Total Ab) H-MC-(D)val-(L)cit- 40 50.4 33.7 3.98 PAB-MMAE (Conj.)
H-MC-(Me)-vc-PAB- 135.1 15.0 44.9 11.2 23.8 MMAE, Total Ab
H-MC-(Me)-vc-PAB- 31.9 63.8 45.2 2.96 MMAE, Conj.
H-MC-(D)val-(D)cit- 88.2 22.8 33.8 10.5 38.3 PAB-MMAE, Total Ab
H-MC-(D)val-(D)cit- 33.6 59.8 36.0 4.43 PAB-MMAE, Conj.
H-MC-vc-PAB-MMAE, 78.6 26.3 39.5 5.8 40.6 Total Ab
H-MC-vc-PAB-MMAE, 31.1 64.4 33.2 3.00 Conj. H linked to MC by
lysine [lys] MMAF 0.99 204 280 0.224 -- 200 .mu.g/kg MMAE 3.71 62.6
649 0.743 -- 206 .mu.g/kg HER F(ab`).sub.2-MC-vc- 9.3 217 34.4 0.35
95 MMAE, Total Ab HER F(ab`).sub.2-MC-vc- 8.8 227 36.9 0.29 MMAE,
Conj. 4D5-H-Fab-MC-vc- 43.8 46.2 38.5 1.49 68 MMAF, Total Ab
4D5-H-Fab-MC-vc- 29.9 68.1 34.1 1.12 MMAF, Conj. 4D5-H-Fab-MC-vc-
71.5 70.3 108 1.18 59 MMAE, Total Ab 4D5-H-Fab-MC-vc- 42.2 118.9
114 0.74 MMAE, Conj. 4D5-H-Fab 93.4 53.9 133 1.08 --
H-MC-vc-PAB-MMAF, 170 12.03 47.9 8.44 49.5 Total Ab
H-MC-vc-PAB-MMAF, 83.9 23.96 44.7 4.01 Conj. H-MC-vc-PAB-MMAF- 211
9.8 39.8 8.53 34.3 DMAEA, Total Ab H-MC-vc-PAB-MMAF- 71.5 28.2 38.8
3.64 DMAEA, Conj. H-MC-vc-PAB-MMAF- 209 9.75 53.2 8.32 29.7 TEG,
Total Ab H-MC-vc-PAB-MMAF- 63.4 31.8 34.9 4.36 TEG, Conj. H =
trastuzumab linked via a cysteine [cys] except where noted 2 mg/kg
dose except where noted
[0915] AUC inf is the area under the plasma concentration-time
curve from time of dosing to infinity and is a measure of the total
exposure to the measured entity (drug, ADC). CL is defined as the
volume of plasma cleared of the measured entity in unit time and is
expressed by normalizing to body weight. T1/2 term is the half-life
of the drug in the body measured during its elimination phase. The
% Conj. term is the relative amount of ADC compared to total
antibody detected, by separate ELISA immunoaffinity tests
("Analytical Methods for Biotechnology Products", Ferraiolo et al,
p85-98 in Pharmacokinetics of Drugs (1994) P. G. Welling and L. P.
Balant, Eds., Handbook of Experimental Pharmacology, Vol. 110,
Springer-Verlag. The % Conj. calculation is simply AUCinf of ADC/
AUCinf total Ab, and is a general indicator of linker stability,
although other factors and mechanisms may be in effect.
[0916] FIG. 11 shows a graph of a plasma concentration clearance
study after administration of the antibody drug conjugates:
H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to Sprague-Dawley rats.
Concentrations of total antibody and ADC were measured over
time.
[0917] FIG. 12 shows a graph of a two stage plasma concentration
clearance study where ADC was administered at different dosages and
concentrations of total antibody and ADC were measured over
time.
In Vivo Efficacy
[0918] The in vivo efficacy of the ADC of the invention was
measured by a high expressing HER2 transgenic explant mouse model.
An allograft was propagated from the Fo5 mmtv transgenic mouse
which does not respond to, or responds poorly to, HERCEPTIN.RTM.
therapy. Subjects were treated once with ADC and monitored over 3-6
weeks to measure the time to tumor doubling, log cell kill, and
tumor shrinkage. Follow up dose-response and multi-dose experiments
were conducted.
[0919] Tumors arise readily in transgenic mice that express a
mutationally activated form of neu, the rat homolog of HER2, but
the HER2 that is overexpressed in breast cancers is not mutated and
tumor formation is much less robust in transgenic mice that
overexpress nonmutated HER2 (Webster et al. (1994) Semin. Cancer
Biol. 5:69-76).
[0920] To improve tumor formation with nonmutated HER2, transgenic
mice were produced using a HER2 cDNA plasmid in which an upstream
ATG was deleted in order to prevent initiation of translation at
such upstream ATG codons, which would otherwise reduce the
frequency of translation initiation from the downstream authentic
initiation codon of HER2 (for example, see Child et al. (1999) J.
Biol. Chem. 274: 24335-24341). Additionally, a chimeric intron was
added to the 5' end, which should also enhance the level of
expression as reported earlier (Neuberger and Williams (1988)
Nucleic Acids Res. 16: 6713; Buchman and Berg (1988) Mol. Cell.
Biol. 8:4395; Brinster et al. (1988) Proc. Natl. Acad. Sci. USA
85:836). The chimeric intron was derived from a Promega vector,
pCI-neo mammalian expression vector (bp 890-1022). The cDNA 3'-end
is flanked by human growth hormone exons 4 and 5, and
polyadenylation sequences. Moreover, FVB mice were used because
this strain is more susceptible to tumor development. The promoter
from MMTV-LTR was used to ensure tissue-specific HER2 expression in
the mammary gland. Animals were fed the AIN 76A diet in order to
increase susceptibility to tumor formation (Rao et al. (1997)
Breast Cancer Res. and Treatment 45:149-158).
TABLE-US-00042 TABLE 2d Tumor measurements in allograft mouse model
- MMTV-HER2 Fo5 Mammary Tumor, athymic nude mice single dose at day
1 (T = 0) except where noted H = trastuzumab linked via a cysteine
[cys] except where noted Tumor Mean doubling log cell Sample time
kill Drugs per antibody Dose Ti PR CR (days) Vehicle 2-5 0
H-MC-vc-PAB-MMAE 1250 .mu.g/m.sup.2 5/5 4/7 0/7 18 1.5 8.7 MMAE/Ab
H-MC-vc-PAB-MMAF 555 .mu.g/m.sup.2 2/5 2/7 5/7 69 6.6 3.8 MMAF/Ab
H-MC(Me)-vc-PAB-MMAF >50 6.4 H-MC-MMAF 4.8 MMAF/Ab 9.2 mg/kg Ab
7/7 6/7 0/7 63 9 550 .mu.g/m.sup.2 at 0, 7, 14 and 21 days
H-MC-MMAF 4.8 MMAF/Ab 14 mg/kg Ab 5/5 5/7 2/7 >63 840
.mu.g/m.sup.2 at 0, 7, 14 and 21 days H-MC-vc-PAB-MMAF 3.5 mg/kg Ab
5/6 1/7 3/7 >36 5.9 MMAF/Ab 300 .mu.g/m.sup.2 at 0, 21, and 42
days H-MC-vc-PAB-MMAF 4.9 mg/kg Ab 4/7 2/7 5/7 >90 5.9 MMAF/Ab
425 .mu.g/m.sup.2 at 0, 21, and 42 days H-MC-vc-PAB-MMAF 6.4
mg/kgAb 3/6 1/7 6/7 >90 5.9 MMAF/Ab 550 .mu.g/m.sup.2 at 0, 21,
and 42 days H-(L)val-(L)cit-MMAE 10 mg/kg 7/7 1/7 0/7 15.2 1.1 8.7
MMAE/Ab H-MC-MMAE 10 mg/kg 7/7 0/7 0/7 4 0.1 4.6 MMAE/Ab
H-(D)val-(D)cit-MMAE 10 mg/kg 7/7 0/7 0/7 3 4.2 MMAE/Ab
H-(D)val-(L)cit-MMAE 13 mg/kg 7/7 0/7 0/7 9 0.6 3.2 MMAE/Ab
H-MC(Me)-vc-MMAE 13 mg/kg 7/7 3/7 0/7 17 1.2 3.0 MMAE/Ab
H-(L)val-(D)cit-MMAE 12 mg/kg 7/7 0/7 0/7 5 0.2 3.5 MMAE/Ab
H-vc-MMAE 10 mg/kg 7/7 17 8.7 MMAE/Ab H-cys-vc-MMAF 1 mg/kg 7/7 3
3.8 MMAF/Ab H-cys-vc-MMAF 3 mg/kg 7/7 >17 3.8 MMAF/Ab
H-cys-vc-MMAF 10 mg/kg 4/7 4/7 3/7 >17 3.8 MMAF/Ab
H-MC-vc-MMAF-TEG 10 mg/kg 3/6 1/7 6/7 81 7.8 4 MMAF/Ab
H-MC-vc-MMAF-TEG 10 mg/kg 0/5 0/7 7/7 81 7.9 4 MMAF/Ab q3wk .times.
3 H-vc-MMAF (lot 1) 10 mg/kg 4/6 2/8 5/8 H-vc-MMAF (lot 2) 10 mg/kg
7/8 1/8 1/8 H-MC-MMAF 10 mg/kg 8/8 1/8 0/8 18 550 .mu.g/m.sup.2
H-(Me)-vc-MMAF 10 mg/kg 3/7 2/8 5/8 H-vc-MMAE 7.5 MMAE/Ab 3.7 mg/kg
at 6/6 0/7 1/7 17 2.3 0, 7, 14, 21, 28 days H-vc-MMAE 7.5 MMAE/Ab
7.5 mg/kg at 5/7 3/7 3/7 69 10 0, 7, 14, 21, 28 days anti
IL8-vc-MMAE 7.5 mg/kg at 7/7 0/7 0/7 5 0.5 7.5 MMAE/Ab 0, 7, 14,
21, 28 days anti IL8-vc-MMAE 3.7 mg/kg at 6/6 0/7 0/7 3 0.2 7.5
MMAE/Ab 0, 7, 14, 21, 28 days H-fk-MMAE 7.5 MMAE/Ab 7.5 mg/kg at
7/7 1/7 0/7 31 4.4 0, 7, 14, 21, 28 days H-fk-MMAE 7.5 MMAE/Ab 3.7
mg/kg at 7/7 0/7 0/7 8.3 0.9 0, 7, 14, 21, 28 days anti IL8-fk-MMAE
7.5 mg/kg at 7/7 0/7 0/7 6 0.5 7.5 MMAE/Ab 0, 7, 14, 21, 28 days
anti IL8-&-MMAE 3.7 mg/kg at 7/7 0/7 0/7 3 0.1 7.5 MMAE/Ab 0,
7, 14, 21, 28 days Trastuzumab 7.5 mg/kg at 7/7 0/7 0/7 5 0.4 0, 7,
14, 21, 28 days H-vc-MMAE 10 mg/kg 6/6 3/6 0/6 15 1.3 8.7 MMAE/Ab
1250 .mu.g/m.sup.2 H-vc-MMAE 10 mg/kg 7/7 5/7 >19 1250
.mu.g/m.sup.2 at 0, 7, and 14 days H-vc-MMAE 3 mg/kg at 0, 7/7 8 7,
and 14 days H-vc-MMAE 1 mg/kg at 0, 7/7 7 7, and 14 days H-vc-MMAF
10 mg/kg 8/8 5/8 >21 H-vc-MMAF 10 mg/kg at 4/7 4/7 3/7 >21 0,
7, and 14 days H-vc-MMAF 3 mg/kg at 0, 7/7 6 7, and 14 days
H-vc-MMAF 1 mg/kg at 0, 8/8 4 7, and 14 days Trastuzumab 10 mg/kg
at 8/8 3 0 and 7 days Hg-MC-vc-PAB-MMAF 10 mg/kg at 6/7 3/8 5/8 56
5.1 4.1 MMAF/Ab 0 days Fc8-MC-vc-PAB-MMAF 10 mg/kg at 7/7 6/8 0/8
25 2.1 4.4 MMAF/Ab 0 days 7C2-MC-vc-PAB-MMAF 10 mg/kg at 5/6 6/8
1/8 41 3.7 4 MMAF/Ab 0 days H-MC-vc-PAB-MMAF 10 mg/kg at 3/8 3/8
5/8 62 5.7 5.9 MMAF/Ab 0 days 2H9-MC-vc-PAB-MMAE 9/9 >14 days
2H9-MC-vc-PAB-MMAF 9/9 >14 days 11D10-vc-PAB-MMAE 9/9 >14
days 11D10-vc-PAB-MMAF 9/9 11 days 7C2 = anti-HER2 murine antibody
which binds a different epitope than trastuzumab. Fc8 = mutant that
does not bind to FcRn Hg = "Hingeless" full-length humanized 4D5,
with heavy chain hinge cysteines mutated to serines. Expressed in
E. coli (therefore non-glycosylated.) 2H9 = Anti-EphB2R 11D10 =
Anti-0772P
[0921] The term Ti is the number of animals in the study group with
tumor at T=0/total animals in group. The term PR is the number of
animals attaining partial remission of tumor/ animals with tumor at
T=0 in group. The term CR is the number of animals attaining
complete remission of tumor/ animals with tumor at T=0 in group.
The term Log cell kill is the time in days for the tumor volume to
double--the time in days for the control tumor volume to double
divided by 3.32.times. time for tumor volume to double in control
animals (dosed with Vehicle). The log-cell-kill calculation takes
into account tumor growth delay resulting from treatment and tumor
volume doubling time of the control group. Anti-tumor activity of
ADC is classified with log-cell-kill values of:
TABLE-US-00043 ++++ .gtoreq.3.4 (highly active) +++ = 2.5-3.4 ++ =
1.7-2.4 + = 1.0-1.6 inactive = 0
[0922] FIG. 13 shows the mean tumor volume change over time in
athymic nude mice with MMTV-HER2Fo5 Mammary tumor allografts dosed
on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250
.mu.g/m.sup.2) and Trastuzumab-MC-vc-PAB-MMAF (555 .mu.g/m.sup.2).
(H=Trastuzumab). The growth of tumors was retarded by treatment
with ADC as compared to control (Vehicle) level of growth. FIG. 14
shows the mean tumor volume change over time in athymic nude mice
with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10
mg/kg (660 .mu.g/m.sup.2) of Trastuzumab-MC-MMAE and 1250
.mu.g/m.sup.2 Trastuzumab-MC-vc-PAB-MMAE. FIG. 15 shows the mean
tumor volume change over time in athymic nude mice with MMTV-HER2
Fo5 Mammary tumor allografts dosed with 650 .mu.g/m.sup.2
Trastuzumab-MC-MMAF. Table 2d and FIGS. 13-15 show that the ADC
have strong anti-tumor activity in the allograft of a HER2 positive
tumor (Fo5) that originally arose in an MMTV-HER2 transgenic mouse.
The antibody alone (e.g., Trastuzumab) does not have significant
anti-tumor activity in this model (Erickson et al. U.S. Pat. No.
6,632,979). As illustrated in FIGS. 13-15, the growth of the tumors
was retarded by treatment with ADC as compared to control (Vehicle)
level of growth.
[0923] In a surprising and unexpected discovery, the in vivo
anti-tumor activity results of the ADC in Table 2d show generally
that ADC with a low average number of drug moieties per antibody
showed efficacy, e.g., tumor doubling time >15 days and mean log
cell kill >1.0. FIG. 16 shows that for the antibody drug
conjugate, trastuzumab-MC-vc-PAB-MMAF, the mean tumor volume
diminished and did not progress where the MMAF:trastuzumab ratio
was 2 and 4, whereas tumor progressed at a ratio of 5.9 and 6, but
at a rate lower than Vehicle (buffer). The rate of tumor
progression in this mouse xenograft model was about the same, i.e.
3 days, for Vehicle and trastuzumab. The results suggest that at
least for trastuzumab ADC, the optimal ratio of drug moieties per
antibody may be less than about 8, and may be about 2 to about
4.
4.5.5 Rodent Toxicity
[0924] Antibody drug conjugates and an ADC-minus control,
"Vehicle", were evaluated in an acute toxicity rat model. Toxicity
of ADC was investigated by treatment of male and female
Sprague-Dawley rats with the ADC and subsequent inspection and
analysis of the effects on various organs. Gross observations
included changes in body weights and signs of lesions and bleeding.
Clinical pathology parameters (serum chemistry and hematology),
histopathology, and necropsy were conducted on dosed animals.
[0925] It is considered that weight loss, or weight change relative
to animals dosed only with Vehicle, in animals after dosing with
ADC is a gross and general indicator of systemic or localized
toxicity. FIGS. 17-19 show the effects of various ADC and control
(Vehicle) after dosing on rat body weight.
[0926] Hepatotoxicity was measured by elevated liver enzymes,
increased numbers of mitotic and apoptotic figures and hepatocyte
necrosis. Hematolymphoid toxicity was observed by depletion of
leukocytes, primarily granuloctyes (neutrophils), and/or platelets,
and lymphoid organ involvement, i.e. atrophy or apoptotic activity.
Toxicity was also noted by gastrointestinal tract lesions such as
increased numbers of mitotic and apoptotic figures and degenerative
enterocolitis.
[0927] Enzymes indicative of liver injury that were studied
include:
[0928] AST (aspartate aminotransferase)
[0929] Localization: cytoplasmic; liver, heart, skeletal muscle,
kidney
[0930] Liver:Plasma ratio of 7000:1
[0931] T1/2: 17 hrs
ALT (alanine aminotransferase)
[0932] Localization: cytoplasmic; liver, kidney, heart, skeletal
muscle
[0933] Liver:Plasma ratio of 3000:1 [0934] T1/2: 42 hrs; diurnal
variation [0935] GGT (g-glutamyl transferase) [0936] Localization:
plasma membrane of cells with high secretory or absorptive
capacity; liver, kidney, intestine [0937] Poor predictor of liver
injury; commonly elevated in bile duct disorders
[0938] The toxicity profiles of trastuzumab-MC-val-cit-MMAF,
trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and
trastuzumab-MC-val-cit-PAB-MMAF were studied in female
Sprague-Dawley rats (Example 19). The humanized trastuzumab
antibody does not bind appreciably to rat tissue, and any toxicity
would be considered non-specific. Variants at dose levels of 840
and 2105 ug/m.sup.2 MMAF were compared to
trastuzumab-MC-val-cit-PAB-MMAF at 2105 ug/m.sup.2.
[0939] Animals in groups 1, 2, 3, 4, 6, and 7 (Vehicle, 9.94 &
24.90 mg/kg trastuzumab-MC-val-cit-MMAF, 10.69 mg/kg
trastuzumab-MC(Me)-val-cit-PAB-MMAF, and 10.17 & 25.50 mg/kg
trastuzumab-MC-MMAF, respectively) gained weight during the study.
Animals in groups 5 and 8 (26.78 mg/kg
trastuzumab-MC(Me)-val-cit-PAB-MMAF and 21.85 mg/kg
trastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight during
the study. On Study Day 5, the change in body weights of animals in
groups 2, 6 and 7 were not significantly different from group 1
animals. The change in body weights of animals in groups 3, 4, 5
and 8 were statistically different from group 1 animals (Example
19).
[0940] Rats treated with trastuzumab-MC-MMAF (groups 6 and 7) were
indistinguishable from vehicle-treated control animals at both dose
levels; i.e. this conjugate showed a superior safety profile in
this model. Rats treated with trastuzumab-MC-val-cit-MMAF (without
the self-immolative PAB moiety; groups 2 and 3) showed
dose-dependent changes typical for MMAF conjugates; the extent of
the changes was less compared with a full length
MC-val-cit-PAB-MMAF conjugate (group 8). The platelet counts on day
5 were at approximately 30% of baseline values in animals of group
3 (high dose trastuzumab-MC-val-cit-MMAF) compared with 15% in
animals of group 8 (high dose trastuzumab-MC-val-cit-PAB-MMAF).
Elevation of liver enzymes AST and ALT, of bilirubin and the extent
of thrombocytopenia was most evident in animals treated with
trastuzumab-MC(Me)-val-cit-PAB-MMAF (groups 4 and 5) in a
dose-dependent fashion; animals of group 5 (high dose group) showed
on day 5 levels of ALT of approximately 10.times. the baseline
value and platelets were reduced by approximately 90% at the time
of necropsy.
[0941] Female Sprague Dawley Rats were also dosed at high levels
(Example 19, High Dose study: Groups 2, 3, 4) with
trastuzumab-MC-MMAF, and Vehicle control (Group 1). Mild toxicity
signals were observed, including a dose-dependent elevation of
liver enzymes ALT, AST and GGT. On day 5 animals in the highest
dose group showed a 2-fold elevation of ALT and a 5-fold elevation
of AST; GGT is also elevated (6 U/L). Enzyme levels show a trend
towards normalization on day 12. There was a mild granulocytosis in
all three dose groups on day 5, the platelet count remained
essentially unchanged in all animals. Morphological changes were
mild; animals treated at the 4210 .mu.g/m.sup.2 dose level (Group
2) showed unremarkable histology of liver, spleen, thymus,
intestines and bone marrow. Mildly increased apoptotic and mitotic
activity was observed in thymus and liver, respectively in animals
treated at the 5500 .mu.g/m.sup.2 dose level (Group 3). The bone
marrow was normocellular, but showed evidence of granulocytic
hyperplasia, which is consistent with the absolute granulocytosis
observed in the peripheral blood counts in these animals. Animals
at the highest dose in group 4 showed qualitatively the same
features; the mitotic activity in the liver appears somewhat
increased compared to animals in Group 3. Also, extramedullary
hematopoiesis was seen in spleen and liver.
[0942] EphB2R is a type 1 TM tyrosine kinase receptor with close
homology between mouse and human, and is over-expressed in
colorectal cancer cells. 2H9 is an antibody against EphB2R. The
naked antibody has no effect on tumor growth, but 2H9-val-cit-MMAE
killed EphB2R expressing cells and showed efficacy in a mouse
xenograft model using CXF1103 human colon tumors (Mao et al (2004)
Cancer Res. 64:781-788). 2H9 and 7C2 are both mouse IgG1 anti-HER2
antibodies. The toxicity profiles of 2H9-MC-val-cit-PAB-MMAF (3.7
MMAF/Ab), 7C2-MC-val-cit-PAB-MMAF (4 MMAF/Ab), and
trastuzumab-MC-val-cit-PAB-MMAF (5.9 MMAF/Ab) were compared. The
differences in the structure of each immunoconjugate or the drug
portion of the immunoconjugate may affect the pharmacokinetics and
ultimately the safety profile. The humanized trastuzumab antibody
does not bind appreciably to rat tissue, and any toxicity would be
considered non-specific.
Cynomolgus Monkey Toxicity/Safety
[0943] Similar to the rat toxicity/safety study, cynomolgus monkeys
were treated with ADC followed by liver enzyme measurements, and
inspection and analysis of the effects on various organs. Gross
observations included changes in body weights and signs of lesions
and bleeding. Clinical pathology parameters (serum chemistry and
hematology), histopathology, and necropsy were conducted on dosed
animals (Example 19).
[0944] The antibody drug conjugate, H-MC-vc-PAB-MMAE (H=trastuzumab
linked through cysteine) showed no evidence of liver toxicity at
any of the dose levels tested. Peripheral blood granulocytes showed
depletion after a single dose of 1100 mg/m.sup.2 with complete
recovery 14 days post-dose. The antibody drug conjugate
H-MC-vc-PAB-MMAF showed elevation of liver enzymes at 550
(transient) and 880 mg/m.sup.2 dose level, no evidence of
granulocytopenia, and a dose-dependent, transient (groups 2 &
3) decline of platelets.
4.6 Synthesis of the Compounds of the Invention
[0945] The Exemplary Compounds and Exemplary Conjugates can be made
using the synthetic procedures outlined below in FIGS. 25-36. As
described in more detail below, the Exemplary Compounds or
Exemplary Conjugates can be conveniently prepared using a Linker
having a reactive site for binding to the Drug and Ligand. In one
aspect, a Linker has a reactive site which has an electrophilic
group that is reactive to a nucleophilic group present on a Ligand,
such as but not limited to an antibody. Useful nucleophilic groups
on an antibody include but are not limited to, sulfhydryl, hydroxyl
and amino groups. The heteroatom of the nucleophilic group of an
antibody is reactive to an electrophilic group on a Linker and
forms a covalent bond to a Linker unit. Useful electrophilic groups
include, but are not limited to, maleimide and haloacetamide
groups. The electrophilic group provides a convenient site for
antibody attachment.
[0946] In another embodiment, a Linker has a reactive site which
has a nucleophilic group that is reactive to an electrophilic group
present on an antibody. Useful electrophilic groups on an antibody
include, but are not limited to, aldehyde and ketone carbonyl
groups. The heteroatom of a nucleophilic group of a Linker can
react with an electrophilic group on an antibody and form a
covalent bond to an antibody unit. Useful nucleophilic groups on a
Linker include, but are not limited to, hydrazide, oxime, amino,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide. The electrophilic group on an antibody provides a
convenient site for attachment to a Linker.
[0947] Carboxylic acid functional groups and chloroformate
functional groups are also useful reactive sites for a Linker
because they can react with secondary amino groups of a Drug to
form an amide linkage. Also useful as a reactive site is a
carbonate functional group on a Linker, such as but not limited to
p-nitrophenyl carbonate, which can react with an amino group of a
Drug, such as but not limited to N-methyl valine, to form a
carbamate linkage. Typically, peptide-based Drugs can be prepared
by forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry.
[0948] The synthesis of an illustrative Stretcher having an
electrophilic maleimide group is illustrated below in FIGS. 28 and
29. General synthetic methods useful for the synthesis of a Linker
are described in FIG. 30. FIG. 31 shows the construction of a
Linker unit having a val-cit group, an electrophilic maleimide
group and a PAB self-immolative Spacer group. FIG. 32 depicts the
synthesis of a Linker having a phe-lys group, an electrophilic
maleimide group, with and without the PAB self-immolative Spacer
group. FIG. 33 presents a general outline for the synthesis of a
Drug-Linker Compound, while FIG. 34 presents an alternate route for
preparing a Drug-Linker Compound. FIG. 35 depicts the synthesis of
a branched linker containing a BHMS group. FIG. 36 outlines the
attachment of an antibody to a Drug-Linker Compound to form a
Drug-Linker-Antibody Conjugate, and FIG. 34 illustrates the
synthesis of Drug-Linker-Antibody Conjugates having, for example
but not limited to, 2 or 4 drugs per Antibody.
[0949] As described in more detail below, the Exemplary Conjugates
are conveniently prepared using a Linker having two or more
Reactive Sites for binding to the Drug and a Ligand. In one aspect,
a Linker has a Reactive site which has an electrophilic group that
is reactive to a nucleophilic group present on a Ligand, such as an
antibody. Useful nucleophilic groups on an antibody include but are
not limited to, sulfhydryl, hydroxyl and amino groups. The
heteroatom of the nucleophilic group of an antibody is reactive to
an electrophilic group on a Linker and forms a covalent bond to a
Linker unit. Useful electrophilic groups include, but are not
limited to, maleimide and haloacetamide groups. The electrophilic
group provides a convenient site for antibody attachment.
[0950] In another embodiment, a Linker has a Reactive site which
has a nucleophilic group that is reactive to an electrophilic group
present on a Ligand, such as an antibody. Useful electrophilic
groups on an antibody include, but are not limited to, aldehyde and
ketone carbonyl groups. The heteroatom of a nucleophilic group of a
Linker can react with an electrophilic group on an antibody and
form a covalent bond to an antibody unit. Useful nucleophilic
groups on a Linker include, but are not limited to, hydrazide,
oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate,
and arylhydrazide. The electrophilic group on an antibody provides
a convenient site for attachment to a Linker.
4.6.1 Drug Moiety Synthesis
[0951] Typically, peptide-based Drugs can be prepared by forming a
peptide bond between two or more amino acids and/or peptide
fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry.
[0952] The auristatin/dolastatin drug moieties may be prepared
according to the general methods of: U.S. Pat. No. 5,635,483; U.S.
Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc.
111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design
13:243-277; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1
5:859-863.
[0953] In one embodiment, a Drug is prepared by combining about a
stoichiometric equivalent of a dipeptide and a tripeptide,
preferably in a one-pot reaction under suitable condensation
conditions. This approach is illustrated in FIGS. 25-27, below.
[0954] FIG. 25 illustrates the synthesis of an N-terminal
tripeptide unit F which is a useful intermediate for the synthesis
of the drug compounds of Formula Ib.
##STR00040##
[0955] As illustrated in FIG. 25, a protected amino acid A (where
PG represents an amine protecting group, R.sup.4 is selected from
hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, alkyl-aryl,
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle,
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from H and methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from hydrogen, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and 6,
and form a ring with the carbon atom to which they are attached) is
coupled to t-butyl ester B (where R.sup.6 is selected from --H and
--C.sub.1-C.sub.8 alkyl; and R.sup.7 is selected from hydrogen,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, alkyl-aryl,
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
alkyl-(C.sub.3-C.sub.8 heterocycle)) under suitable coupling
conditions, e.g., in the presence of PyBrop and
diisopropylethylamine, or using DCC (see, for example, Miyazaki, K.
et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).
[0956] Suitable protecting groups PG, and suitable synthetic
methods to protect an amino group with a protecting group are well
known in the art. See, e.g., Greene, T. W. and Wuts, P.G.M.,
Protective Groups in Organic Synthesis, 2nd Edition, 1991, John
Wiley & Sons. Exemplary protected amino acids A are PG-Ile and,
particularly, PG-Val, while other suitable protected amino acids
include, without limitation: PG-cyclohexylglycine,
PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid,
PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and
PG-tert-butylglycine. Z is an exemplary protecting group. Fmoc is
another exemplary protecting group. An exemplary t-butyl ester B is
dolaisoleuine t-butyl ester.
[0957] The dipeptide C can be purified, e.g., using chromatography,
and subsequently deprotected, e.g., using H.sub.2 and 10% Pd--C in
ethanol when PG is benzyloxycarbonyl, or using diethylamine for
removal of an Fmoc protecting group. The resulting amine D readily
forms a peptide bond with an amino acid BB (wherein R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; and R.sup.3 is selected from hydrogen, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, alkyl-aryl, alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and alkyl-(C.sub.3-C.sub.8
heterocycle)). N,N-Dialkyl amino acids are exemplary amino acids
for BB, such as commercially available N,N-dimethyl valine. Other
N,N-dialkyl amino acids can be prepared by reductive bis-alkylation
using known procedures (see, e.g., Bowman, R. E, Stroud, H. H J.
Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine
are two exemplary amino acids BB useful for the synthesis of
N-monoalkyl derivatives. The amine D and the amino acid BB react to
provide the tripeptide E using coupling reagent DEPC with
triethylamine as the base. The C-terminus protecting group of E is
subsequently deprotected using HCl to provide the tripeptide
compound of formula F.
[0958] Illustrative DEPC coupling methodology and the PyBrop
coupling methodology shown in FIG. 25 are outlined below in General
Procedure A and General Procedure B, respectively. Illustrative
methodology for the deprotection of a Z-protected amine via
catalytic hydrogenation is outlined below in General Procedure
C.
[0959] General Procedure A: Peptide synthesis using DEPC. The
N-protected or N,N-disubstituted amino acid or peptide D (1.0 eq.)
and an amine BB (1.1 eq.) are diluted with an aprotic organic
solvent, such as dichloromethane (0.1 to 0.5 M). An organic base
such as triethylamine or diisopropylethylamine (1.5 eq.) is then
added, followed by DEPC (1.1 eq.). The resulting solution is
stirred, preferably under argon, for up to 12 hours while being
monitored by HPLC or TLC. The solvent is removed in vacuo at room
temperature, and the crude product is purified using, for example,
HPLC or flash column chromatography (silica gel column). Relevant
fractions are combined and concentrated in vacuo to afford
tripeptide E which is dried under vacuum overnight.
[0960] General procedure B: Peptide synthesis using PyBrop. The
amino acid B (1.0 eq.), optionally having a carboxyl protecting
group, is diluted with an aprotic organic solvent such as
dichloromethane or DME to provide a solution of a concentration
between 0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is
added. Fmoc-, or Z-protected amino acid A (1.1 eq.) is added as a
solid in one portion, then PyBrop (1.2 eq.) is added to the
resulting mixture. The reaction is monitored by TLC or HPLC,
followed by a workup procedure similar to that described in General
Procedure A.
[0961] General procedure C: Z-removal via catalytic hydrogenation.
Z-protected amino acid or peptide C is diluted with ethanol to
provide a solution of a concentration between 0.5 and 1.0 mM in a
suitable vessel, such as a thick-walled round bottom flask. 10%
palladium on carbon is added (5-10% w/w) and the reaction mixture
is placed under a hydrogen atmosphere. Reaction progress is
monitored using HPLC and is generally complete within 1-2 h. The
reaction mixture is filtered through a pre-washed pad of celite and
the celite is again washed with a polar organic solvent, such as
methanol after filtration. The eluent solution is concentrated in
vacuo to afford a residue which is diluted with an organic solvent,
preferably toluene. The organic solvent is then removed in vacuo to
afford the deprotected amine C.
[0962] FIG. 26 shows a method useful for making a C-terminal
dipeptide of formula K and a method for coupling the dipeptide of
formula K with the tripeptide of formula F to make drug compounds
of Formula Ib.
##STR00041##
[0963] The dipeptide K can be readily prepared by condensation of
the modified amino acid Boc-Dolaproine G (see, for example, Pettit,
G. R., et al. Synthesis, 1996, 719-725), with an amine of formula H
using condensing agents well known for peptide chemistry, such as,
for example, DEPC in the presence of triethylamine, as shown in
FIG. 25.
[0964] The dipeptide of formula K can then be coupled with a
tripeptide of formula F using General Procedure D to make the
Fmoc-protected drug compounds of formula L which can be
subsequently deprotected using General Procedure E in order to
provide the drug compounds of formula (Ib).
[0965] General procedure D: Drug synthesis. A mixture of dipeptide
K (1.0 eq.) and tripeptide F (1 eq.) is diluted with an aprotic
organic solvent, such as dichloromethane, to form a 0.1M solution,
then a strong acid, such as trifluoroacetic acid (1/2 v/v) is added
and the resulting mixture is stirred under a nitrogen atmosphere
for two hours at 0.degree. C. The reaction can be monitored using
TLC or, preferably, HPLC. The solvent is removed in vacuo and the
resulting residue is azeotropically dried twice, preferably using
toluene. The resulting residue is dried under high vacuum for 12 h
and then diluted with and aprotic organic solvent, such as
dichloromethane. An organic base such as triethylamine or
diisopropylethylamine (1.5 eq.) is then added, followed by either
PyBrop (1.2 eq.) or DEPC (1.2 eq.) depending on the chemical
functionality on the residue. The reaction mixture is monitored by
either TLC or HPLC and upon completion, the reaction is subjected
to a workup procedure similar or identical to that described in
General Procedure A.
[0966] General procedure E: Fmoc-removal using diethylamine. An
Fmoc-protected Drug L is diluted with an aprotic organic solvent
such as dichloromethane and to the resulting solution is added
diethylamine (1/2 v/v). Reaction progress is monitored by TLC or
HPLC and is typically complete within 2 h. The reaction mixture is
concentrated in vacuo and the resulting residue is azeotropically
dried, preferably using toluene, then dried under high vacuum to
afford Drug Ib having a deprotected amino group.
[0967] FIG. 27 shows a method useful for making MMAF derivatives of
Formula (Ib).
##STR00042##
[0968] The dipeptide O can be readily prepared by condensation of
the modified amino acid Boc-Dolaproine G (see, for example, Pettit,
G. R., et al. Synthesis, 1996, 719-725), with a protected amino
acid of formula M using condensing agents well known for peptide
chemistry, such as, for example, DEPC in the presence of
triethylamine, as shown in FIGS. 25 and 26.
[0969] The dipeptide of formula O can then be coupled with a
tripeptide of formula F using General Procedure D to make the
Fmoc-protected MMAF compounds of formula P which can be
subsequently deprotected using General Procedure E in order to
provide the MMAF drug compounds of formula (Ib).
[0970] Thus, the above methods are useful for making Drugs that can
be used in the present invention.
4.6.2 Drug Linker Synthesis
[0971] To prepare a Drug-Linker Compound of the present invention,
the Drug is reacted with a reactive site on the Linker. In general,
the Linker can have the structure:
##STR00043##
when both a Spacer unit (-Y-) and a Stretcher unit (-A-) are
present. Alternately, the Linker can have the structure:
##STR00044##
when the Spacer unit (-Y-) is absent.
[0972] The Linker can also have the structure:
##STR00045##
when both the Stretcher unit (-A-) and the Spacer unit (-Y-) are
absent.
[0973] The Linker can also have the structure:
##STR00046##
when both the Amino Acid unit (W) and the Spacer Unit (Y) are
absent.
[0974] In general, a suitable Linker has an Amino Acid unit linked
to an optional Stretcher Unit and an optional Spacer Unit. Reactive
Site 1 is present at the terminus of the Spacer and Reactive site 2
is present at the terminus of the Stretcher. If a Spacer unit is
not present, then Reactive site 1 is present at the C-terminus of
the Amino Acid unit.
[0975] In an exemplary embodiment of the invention, Reactive Site
No. 1 is reactive to a nitrogen atom of the Drug, and Reactive Site
No. 2 is reactive to a sulfhydryl group on the Ligand. Reactive
Sites 1 and 2 can be reactive to different functional groups.
[0976] In one aspect of the invention, Reactive Site No. 1 is
##STR00047##
[0977] In another aspect of the invention, Reactive Site No. 1
is
##STR00048##
[0978] In still another aspect of the invention, Reactive Site No.
1 is a p-nitrophenyl carbonate having the formula
##STR00049##
[0979] In one aspect of the invention, Reactive Site No. 2 is a
thiol-accepting group. Suitable thiol-accepting groups include
haloacetamide groups having the formula
##STR00050##
[0980] wherein X represents a leaving group, preferably O-mesyl,
O-tosyl, --Cl, --Br, or --I; or a maleimide group having the
formula
##STR00051##
[0981] Useful Linkers can be obtained via commercial sources, such
as Molecular Biosciences Inc. (Boulder, Colo.), or prepared as
summarized in FIGS. 28-30.
##STR00052##
wherein X is --CH.sub.2-- or --CH.sub.2OCH.sub.2--; and n is an
integer ranging either from 0-10 when X is --CH.sub.2--; or 1-10
when X is --CH.sub.2OCH.sub.2--.
[0982] The method shown in FIG. 29 combines maleimide with a glycol
under Mitsunobu conditions to make a polyethylene glycol maleimide
Stretcher (see for example, Walker, M. A. J. Org. Chem. 1995, 60,
5352-5), followed by installation of a p-nitrophenyl carbonate
Reactive Site group.
##STR00053##
wherein E is --CH.sub.2-- or --CH.sub.2OCH.sub.2--; and e is an
integer ranging from 0-8;
[0983] Alternatively, PEG-maleimide and PEG-haloacetamide
stretchers can be prepared as described by Frisch, et al.,
Bioconjugate Chem. 1996, 7, 180-186. FIG. 30 illustrates a general
synthesis of an illustrative Linker unit containing a maleimide
Stretcher group and optionally a p-aminobenzyl ether
self-immolative Spacer.
##STR00054##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and n
is an integer ranging from 0-10.
[0984] Useful Stretchers may be incorporated into a Linker using
the commercially available intermediates from Molecular Biosciences
(Boulder, Colo.) described below by utilizing known techniques of
organic synthesis.
[0985] Stretchers of formula (Ma) can be introduced into a Linker
by reacting the following intermediates with the N-terminus of an
Amino Acid unit as depicted in FIGS. 31 and 32:
##STR00055##
[0986] where n is an integer ranging from 1-10 and T is --H or
--SO.sub.3Na;
##STR00056##
[0987] where n is an integer ranging from 0-3;
##STR00057##
[0988] Stretcher units of formula (Mb) can be introduced into a
Linker by reacting the following intermediates with the N-terminus
of an Amino Acid unit:
##STR00058##
[0989] Stretcher units of formula (IV) can be introduced into a
Linker by reacting the following intermediates with the N-terminus
of an Amino Acid unit:
##STR00059##
[0990] Stretcher units of formula (Va) can be introduced into a
Linker by reacting the following intermediates with the N-terminus
of an Amino Acid unit:
##STR00060##
[0991] Other useful Stretchers may be synthesized according to
known procedures. Aminooxy Stretchers of the formula shown below
can be prepared by treating alkyl halides with N-Boc-hydroxylamine
according to procedures described in Jones, D. S. et al.,
Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C. et al.,
Tetrahedron, 1967, 23(11), 4441-4447.
##STR00061##
wherein --R.sup.17-- is selected from --C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-,
-arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 carbocyclo)-, --(C.sub.3-C.sub.8
carbocyclo)-C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
heterocyclo-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10; Isothiocyanate Stretchers of the formula shown
below may be prepared from isothiocyanatocarboxylic acid chlorides
as described in Angew. Chem., 1975, 87(14):517.
##STR00062##
[0992] wherein --R.sup.17-- is as described herein.
[0993] FIG. 30 shows a method for obtaining of a val-cit dipeptide
Linker having a maleimide Stretcher and optionally a p-aminobenzyl
self-immolative Spacer.
##STR00063##
[0994] wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; and m is an integer ranging
from 0-4.
[0995] FIG. 32 illustrates the synthesis of a phe-lys(Mtr)
dipeptide Linker unit having a maleimide Stretcher unit and a
p-aminobenzyl self-immolative Spacer unit. Starting material AD
(lys(Mtr)) is commercially available (Bachem, Torrance, Calif.) or
can be prepared according to Dubowchik, et al. Tetrahedron Letters
(1997) 38:5257-60.
##STR00064##
[0996] wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; and m is an integer ranging
from 0-4.
[0997] As shown in FIG. 33, a Linker can be reacted with an amino
group of a Drug Compound of Formula (Ib) to form a Drug-Linker
Compound that contains an amide or carbamate group, linking the
Drug unit to the Linker unit. When Reactive Site No. 1 is a
carboxylic acid group, as in Linker AJ, the coupling reaction can
be performed using HATU or PyBrop and an appropriate amine base,
resulting in a Drug-Linker Compound AK, containing an amide bond
between the Drug unit and the Linker unit. When Reactive Site No. 1
is a carbonate, as in Linker AL, the Linker can be coupled to the
Drug using HOBt in a mixture of DMF/pyridine to provide a
Drug-Linker Compound AM, containing a carbamate bond between the
Drug unit and the Linker unit
[0998] Alternately, when Reactive Site No. 1 is a good leaving
group, such as in Linker AN, the Linker can be coupled with an
amine group of a Drug via a nucleophilic substitution process to
provide a Drug-Linker Compound having an amine linkage (AO) between
the Drug unit and the Linker unit.
[0999] Illustrative methods useful for linking a Drug to a Ligand
to form a Drug-Linker Compound are depicted in FIG. 33 and are
outlined in General Procedures G-H.
##STR00065##
[1000] General Procedure G: Amide formation using HATU. A Drug (Ib)
(1.0 eq.) and an N-protected Linker containing a carboxylic acid
Reactive site (1.0 eq.) are diluted with a suitable organic
solvent, such as dichloromethane, and the resulting solution is
treated with HATU (1.5 eq.) and an organic base, preferably
pyridine (1.5 eq.). The reaction mixture is allowed to stir under
an inert atmosphere, preferably argon, for 6 h, during which time
the reaction mixture is monitored using HPLC. The reaction mixture
is concentrated and the resulting residue is purified using HPLC to
yield the amide of formula AK.
[1001] Procedure H: Carbamate formation using HOBt. A mixture of a
Linker AL having a p-nitrophenyl carbonate Reactive site (1.1 eq.)
and Drug (Ib) (1.0 eq.) are diluted with an aprotic organic
solvent, such as DMF, to provide a solution having a concentration
of 50-100 mM, and the resulting solution is treated with HOBt (2.0
eq.) and placed under an inert atmosphere, preferably argon. The
reaction mixture is allowed to stir for 15 min, then an organic
base, such as pyridine (1/4 v/v), is added and the reaction
progress is monitored using HPLC. The Linker is typically consumed
within 16 h. The reaction mixture is then concentrated in vacuo and
the resulting residue is purified using, for example, HPLC to yield
the carbamate AM.
[1002] An alternate method of preparing Drug-Linker Compounds is
outlined in FIG. 34. Using the method of FIG. 34, the Drug is
attached to a partial Linker unit (ZA, for example), which does not
have a Stretcher unit attached. This provides intermediate AP,
which has an Amino Acid unit having an Fmoc-protected N-terminus.
The Fmoc group is then removed and the resulting amine intermediate
AQ is then attached to a Stretcher unit via a coupling reaction
catalyzed using PyBrop or DEPC. The construction of Drug-Linker
Compounds containing either a bromoacetamide Stretcher AR or a PEG
maleimide Stretcher AS is illustrated in FIG. 34.
##STR00066##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro or -cyano; and m is an integer ranging from
0-4.
[1003] Methodology useful for the preparation of a Linker unit
containing a branched spacer is shown in FIG. 35.
##STR00067##
[1004] FIG. 35 illustrates the synthesis of a val-cit dipeptide
linker having a maleimide Stretcher unit and a
bis(4-hydroxymethyl)styrene (BHMS) unit. The synthesis of the BHMS
intermediate (AW) has been improved from previous literature
procedures (see International Publication No, WO 9813059 to
Firestone et al., and Crozet, M. P.; Archaimbault, G.; Vanelle, P.;
Nouguier, R. Tetrahedron Lett. (1985) 26:5133-5134) and utilizes as
starting materials, commercially available diethyl
(4-nitrobenzyl)phosphonate (AT) and commercially available
2,2-dimethyl-1,3-dioxan-5-one (AU). Linkers AY and BA can be
prepared from intermediate AW using the methodology described in
FIG. 29.
4.6.3 Dendritic Linkers
[1005] The linker may be a dendritic type linker for covalent
attachment of more than one drug moiety through a branching,
multifunctional linker moiety to a Ligand, such as but not limited
to an antibody (Sun et al. (2002) Bioorganic & Medicinal
Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic &
Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase
the molar ratio of drug to antibody, i.e. loading, which is related
to the potency of the Drug-Linker-Ligand Conjugate. Thus, where a
cysteine engineered antibody bears only one reactive cytsteine
thiol group, a multitude of drug moieties may be attached through a
dendritic linker.
[1006] The following exemplary embodiments of dendritic linker
reagents allow up to nine nucleophilic drug moiety reagents to be
conjugated by reaction with the chloroethyl nitrogen mustard
functional groups:
##STR00068##
4.6.4 Conjugation of Drug Moieties to Antibodies
[1007] FIG. 36 illustrates methodology useful for making
Drug-Linker-Ligand conjugates having about 2 to about 4 drugs per
antibody. An antibody is treated with a reducing agent, such as
dithiothreitol (DTT) to reduce some or all of the cysteine
disulfide residues to form highly nucleophilic cysteine thiol
groups (--CH.sub.2SH). The partially reduced antibody thus reacts
with drug-linker compounds, or linker reagents, with electrophilic
functional groups such as maleimide or .alpha.-halo carbonyl,
according to the conjugation method at page 766 of Klussman, et al.
(2004), Bioconjugate Chemistry 15(4):765-773.
##STR00069##
For example, an antibody, e.g., AC10, dissolved in 500 mM sodium
borate and 500 mM sodium chloride at pH 8.0 is treated with an
excess of 100 mM dithiothreitol (DTT). After incubation at
37.degree. C. for about 30 minutes, the buffer is exchanged by
elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA.
The thiol/Ab value is checked by determining the reduced antibody
concentration from the absorbance at 280 nm of the solution and the
thiol concentration by reaction with DTNB (Aldrich, Milwaukee,
Wis.) and determination of the absorbance at 412 nm. The reduced
antibody dissolved in PBS is chilled on ice. The drug linker, e.g.,
MC-val-cit-PAB-MMAE in DMSO, dissolved in acetonitrile and water at
known concentration, is added to the chilled reduced antibody in
PBS. After about one hour, an excess of maleimide is added to
quench the reaction and cap any unreacted antibody thiol groups.
The reaction mixture is concentrated by centrifugal ultrafiltration
and the ADC, e.g., AC10-MC-vc-PAB-MMAE, is purified and desalted by
elution through G25 resin in PBS, filtered through 0.2 .mu.m
filters under sterile conditions, and frozen for storage.
[1008] A variety of antibody drug conjugates (ADC) were prepared,
with a variety of linkers, and the drug moieties, MMAE and MMAF.
The following table is an exemplary group of ADC which were
prepared following the protocol of Example 27, and characterized by
HPLC and drug loading assay.
TABLE-US-00044 Target isolated amount drug/Ab (antigen) ADC (mg)
ratio 0772P 16E12-MC-vc-PAB-MMAE 1.75 4 0772P 11D10-MC-vc-PAB-MMAE
46.8 4.4 0772P 11D10-MC-vc-PAB-MMAF 54.5 3.8 Brevican
Brevican-MC-MMAF 2 6 Brevican Brevican-MC-vc-MMAF 2 6 Brevican
Brevican-MC-vc-PAB-MMAF 1.4 6 CD21 CD21-MC-vc-PAB-MMAE 38.1 4.3
CD21 CD21-MC-vc-PAB-MMAF 43 4.1 CRIPTO 11F4-MC-vc-PAB-MMAF 6 4.8
CRIPTO 25G8-MC-vc-PAB-MMAF 7.4 4.7 E16 12G12-MC-vc-PAB-MMAE 2.3 4.6
E16 3B5-MC-vc-PAB-MMAE 2.9 4.6 E16 12B9-MC-vc-PAB-MMAE 1.4 3.8 E16
12B9-MC-vc-PAB-MMAE 5.1 4 E16 12G12-MC-vc-PAB-MMAE 3 4.6 E16
3B5-MC-vc-PAB-MMAE 4.8 4.1 E16 3B5-MC-vc-PAB-MMAF 24.7 4.4 EphB2R
2H9-MC-vc-PAB-MMAE 29.9 7.1 EphB2R 2H9-MC-fk-PAB-MMAE 25 7.5 EphB2R
2H9-MC-vc-PAB-MMAE 175 4.1 EphB2R 2H9-MC-vc-PAB-MMAF 150 3.8 EphB2R
2H9-MC-vc-PAB-MMAF 120 3.7 EphB2R 2H9-MC-vc-PAB-MMAE 10.7 4.4
IL-20Ra IL20Ra-fk-MMAE 26 6.7 IL-20Ra IL20Ra-vc-MMAE 27 7.3 EphB2
IL8-MC-vc-PAB-MMAE 251 3.7 MDP MDP-vc-MMAE 32 MPF 19C3-vc-MMAE 1.44
6.5 MPF 7D9-vc-MMAE 4.3 3.8 MPF 19C3-vc-MMAE 7.9 3 MPF
7D9-MC-vc-PAB-MMAF 5 4.3 Napi3b 10H1-vc-MMAE 4.5 4.6 Napi3b
4C9-vc-MMAE 3.0 5.4 Napi3b 10H1-vc-MMAE 4.5 4.8 Napi3b 10H1-vc-MMAF
6.5 4 NCA 3E6-MC-fk-PAB-MMAE 49.6 5.4 NCA 3E6-MC-vc-PAB-MMAE 56.2
6.4 PSCA PSCA-fk-MMAE 51.7 8.9 PSCA PSCA-vc-MMAE 61.1 8.6 Napi3b
10H1-MC-vc-PAB-MMAE 75 4.2 Napi3b 10H1-MC-vc-PAB-MMAF 95 4.4 Napi3b
10H1-MC-MMAF 92 4 EphB2R 2H9-MC-vc-PAB-MMAE 79 5 EphB2R 2H9-MC-MMAF
92 4.9 0772P 11D10(Fc chimera)-MC-vc-PAB- 79 4.3 MMAE 0772P
11D10(Fc chimera)-MC-vc-PAB- 70 4.5 MMAF 0772P 11D10(Fc
chimera)-MC-MMAF 23 4.5 Brevican 6D2-MC-vc-PAB-MMAF 0.3 4.5
Brevican 6D2-MC-MMAF 0.36 4.5 EphB2R 2H9(Fc chimera)-MC-vc-PAB-
1983 4.3 MMAE E16 12B9-MC-vc-PAB-MMAE 14.1 4.6 E16
12B9-MC-vc-PAB-MMAF 16.4 4.5 E16 12G12-MC-vc-PAB-MMAE 10.5 4.1 E16
12G12-MC-vc-PAB-MMAF 10.2 3.8 E16 3B5-MC-vc-PAB-MMAE 58.6 3.8 E16
3B5-MC-vc-PAB-MMAF 8 3.1 0772P 11D10(Fc chimera)-MC-vc-PAB- 340 3.9
MMAE Steap1 (Steap1-92)-MC-vc-PAB-MMAE 3.5 4 Steap1
(Steap1-92)-MC-vc-PAB-MMAF 4.7 4 Steap1 (Steap1-120)-MC-vc-PAB- 2 4
MMAE Steap1 (Steap1-120)-MC-vc-PAB-MMAF 2.3 4 E16
3B5-MC-vc-PAB-MMAF 52.2 4.5
4.7 Compositions and Methods of Administration
[1009] In other embodiments, described is a composition including
an effective amount of an Exemplary Compound and/or Exemplary
Conjugate and a pharmaceutically acceptable carrier or vehicle. For
convenience, the Drug units and Drug-Linker Compounds can be
referred to as Exemplary Compounds, while Drug-Ligand Conjugates
and Drug-Linker-Ligand Conjugates can be referred to as Exemplary
Conjugates. The compositions are suitable for veterinary or human
administration.
[1010] The present compositions can be in any form that allows for
the composition to be administered to a patient. For example, the
composition can be in the form of a solid, liquid or gas (aerosol).
Typical routes of administration include, without limitation, oral,
topical, parenteral, sublingual, rectal, vaginal, ocular,
intra-tumor, and intranasal. Parenteral administration includes
subcutaneous injections, intravenous, intramuscular, intrasternal
injection or infusion techniques. In one aspect, the compositions
are administered parenterally. In yet another aspect, the Exemplary
Compounds and/or the Exemplary Conjugates or compositions are
administered intravenously.
[1011] Pharmaceutical compositions can be formulated so as to allow
an Exemplary Compound and/or Exemplary Conjugate to be bioavailable
upon administration of the composition to a patient. Compositions
can take the form of one or more dosage units, where for example, a
tablet can be a single dosage unit, and a container of an Exemplary
Compound and/or Exemplary Conjugate in aerosol form can hold a
plurality of dosage units.
[1012] Materials used in preparing the pharmaceutical compositions
can be non-toxic in the amounts used. It will be evident to those
of ordinary skill in the art that the optimal dosage of the active
ingredient(s) in the pharmaceutical composition will depend on a
variety of factors. Relevant factors include, without limitation,
the type of animal (e.g., human), the particular form of the
Exemplary Compound or Exemplary Conjugate, the manner of
administration, and the composition employed.
[1013] The pharmaceutically acceptable carrier or vehicle can be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) can be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) can be gaseous or particulate, so as to
provide an aerosol composition useful in, e.g., inhalatory
administration.
[1014] When intended for oral administration, the composition is
preferably in solid or liquid foam, where semi-solid, semi-liquid,
suspension and gel foams are included within the fauns considered
herein as either solid or liquid.
[1015] As a solid composition for oral administration, the
composition can be formulated into a powder, granule, compressed
tablet, pill, capsule, chewing gum, wafer or the like form. Such a
solid composition typically contains one or more inert diluents. In
addition, one or more of the following can be present: binders such
as carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins, disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon
dioxide; sweetening agents such as sucrose or saccharin, a
flavoring agent such as peppermint, methyl salicylate or orange
flavoring, and a coloring agent.
[1016] When the composition is in the form of a capsule, e.g., a
gelatin capsule, it can contain, in addition to materials of the
above type, a liquid carrier such as polyethylene glycol,
cyclodextrin or a fatty oil.
[1017] The composition can be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid can be
useful for oral administration or for delivery by injection. When
intended for oral administration, a composition can comprise one or
more of a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition for administration by injection, one or
more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent can
also be included.
[1018] The liquid compositions, whether they are solutions,
suspensions or other like form, can also include one or more of the
following: sterile diluents such as water for injection, saline
solution, preferably physiological saline, Ringer's solution,
isotonic sodium chloride, fixed oils such as synthetic mono or
digylcerides which can serve as the solvent or suspending medium,
polyethylene glycols, glycerin, cyclodextrin, propylene glycol or
other solvents; antibacterial agents such as benzyl alcohol or
methyl paraben; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
A parenteral composition can be enclosed in ampoule, a disposable
syringe or a multiple-dose vial made of glass, plastic or other
material. Physiological saline is an exemplary adjuvant. An
injectable composition is preferably sterile.
[1019] The amount of the Exemplary Compound and/or Exemplary
Conjugate that is effective in the treatment of a particular
disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques.
In addition, in vitro or in vivo assays can optionally be employed
to help identify optimal dosage ranges. The precise dose to be
employed in the compositions will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances.
[1020] The compositions comprise an effective amount of an
Exemplary Compound and/or Exemplary Conjugate such that a suitable
dosage will be obtained. Typically, this amount is at least about
0.01% of an Exemplary Compound and/or Exemplary Conjugate by weight
of the composition. When intended for oral administration, this
amount can be varied to range from about 0.1% to about 80% by
weight of the composition. In one aspect, oral compositions can
comprise from about 4% to about 50% of the Exemplary Compound
and/or Exemplary Conjugate by weight of the composition. In yet
another aspect, present compositions are prepared so that a
parenteral dosage unit contains from about 0.01% to about 2% by
weight of the Exemplary Compound and/or Exemplary Conjugate.
[1021] For intravenous administration, the composition can comprise
from about 0.01 to about 100 mg of an Exemplary Compound and/or
Exemplary Conjugate per kg of the animal's body weight. In one
aspect, the composition can include from about 1 to about 100 mg of
an Exemplary Compound and/or Exemplary Conjugate per kg of the
animal's body weight. In another aspect, the amount administered
will be in the range from about 0.1 to about 25 mg/kg of body
weight of the Exemplary Compound and/or Exemplary Conjugate.
[1022] Generally, the dosage of an Exemplary Compound and/or
Exemplary Conjugate administered to a patient is typically about
0.01 mg/kg to about 2000 mg/kg of the animal's body weight. In one
aspect, the dosage administered to a patient is between about 0.01
mg/kg to about 10 mg/kg of the animal's body weight, in another
aspect, the dosage administered to a patient is between about 0.1
mg/kg and about 250 mg/kg of the animal's body weight, in yet
another aspect, the dosage administered to a patient is between
about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, in
yet another aspect the dosage administered is between about 0.1
mg/kg to about 10 mg/kg of the animal's body weight, and in yet
another aspect, the dosage administered is between about 1 mg/kg to
about 10 mg/kg of the animal's body weight.
[1023] The Exemplary Compounds and/or Exemplary Conjugate or
compositions can be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.). Administration can be systemic or local.
Various delivery systems are known, e.g., encapsulation in
liposomes, microparticles, microcapsules, capsules, etc., and can
be used to administer an Exemplary Compound and/or Exemplary
Conjugate or composition. In certain embodiments, more than one
Exemplary Compound and/or Exemplary Conjugate or composition is
administered to a patient.
[1024] In specific embodiments, it can be desirable to administer
one or more Exemplary Compounds and/or Exemplary Conjugate or
compositions locally to the area in need of treatment. This can be
achieved, for example, and not by way of limitation, by local
infusion during surgery; topical application, e.g., in conjunction
with a wound dressing after surgery; by injection; by means of a
catheter; by means of a suppository; or by means of an implant, the
implant being of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, or fibers. In one
embodiment, administration can be by direct injection at the site
(or former site) of a cancer, tumor or neoplastic or pre-neoplastic
tissue. In another embodiment, administration can be by direct
injection at the site (or former site) of a manifestation of an
autoimmune disease.
[1025] In certain embodiments, it can be desirable to introduce one
or more Exemplary Compounds and/or Exemplary Conjugate or
compositions into the central nervous system by any suitable route,
including intraventricular and intrathecal injection.
Intraventricular injection can be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[1026] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent, or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant.
[1027] In yet another embodiment, the Exemplary Compounds and/or
Exemplary Conjugate or compositions can be delivered in a
controlled release system, such as but not limited to, a pump or
various polymeric materials can be used. In yet another embodiment,
a controlled-release system can be placed in proximity of the
target of the Exemplary Compounds and/or Exemplary Conjugate or
compositions, e.g., the brain, thus requiring only a fraction of
the systemic dose (see, e.g., Goodson, in Medical Applications of
Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other
controlled-release systems discussed in the review by Langer
(Science 249:1527-1533 (1990)) can be used.
[1028] The term "carrier" refers to a diluent, adjuvant or
excipient, with which an Exemplary Compound and/or Exemplary
Conjugate is administered. Such pharmaceutical carriers can be
liquids, such as water and oils, including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, sesame oil and the like. The carriers can be
saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal
silica, urea, and the like. In addition, auxiliary, stabilizing,
thickening, lubricating and coloring agents can be used. In one
embodiment, when administered to a patient, the Exemplary Compound
and/or Exemplary Conjugate or compositions and pharmaceutically
acceptable carriers are sterile. Water is an exemplary carrier when
the Exemplary Compounds and/or Exemplary Conjugates are
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers also include excipients such as starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
present compositions, if desired, can also contain minor amounts of
wetting or emulsifying agents, or pH buffering agents.
[1029] The present compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, aerosols, sprays, suspensions, or any
other form suitable for use. Other examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[1030] In an embodiment, the Exemplary Compounds and/or Exemplary
Conjugates are formulated in accordance with routine procedures as
a pharmaceutical composition adapted for intravenous administration
to animals, particularly human beings. Typically, the carriers or
vehicles for intravenous administration are sterile isotonic
aqueous buffer solutions. Where necessary, the compositions can
also include a solubilizing agent. Compositions for intravenous
administration can optionally comprise a local anesthetic such as
lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
an Exemplary Compound and/or Exemplary Conjugate is to be
administered by infusion, it can be dispensed, for example, with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the Exemplary Compound and/or Exemplary Conjugate is
administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients can be
mixed prior to administration.
[1031] Compositions for oral delivery can be in the form of
tablets, lozenges, aqueous or oily suspensions, granules, powders,
emulsions, capsules, syrups, or elixirs, for example. Orally
administered compositions can contain one or more optionally
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents such as peppermint, oil of
wintergreen, or cherry; coloring agents; and preserving agents, to
provide a pharmaceutically palatable preparation. Moreover, where
in tablet or pill form, the compositions can be coated to delay
disintegration and absorption in the gastrointestinal tract thereby
providing a sustained action over an extended period of time.
Selectively permeable membranes surrounding an osmotically active
driving compound are also suitable for orally administered
compounds. In these later platforms, fluid from the environment
surrounding the capsule is imbibed by the driving compound, which
swells to displace the agent or agent composition through an
aperture. These delivery platforms can provide an essentially zero
order delivery profile as opposed to the spiked profiles of
immediate release formulations. A time-delay material such as
glycerol monostearate or glycerol stearate can also be used.
[1032] The compositions can be intended for topical administration,
in which case the carrier may be in the form of a solution,
emulsion, ointment or gel base. If intended for transdermal
administration, the composition can be in the form of a transdermal
patch or an iontophoresis device. Topical formulations can comprise
a concentration of an Exemplary Compound and/or Exemplary Conjugate
of from about 0.05% to about 50% w/v (weight per unit volume of
composition), in another aspect, from 0.1% to 10% w/v.
[1033] The composition can be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the Exemplary Compound and/or Exemplary Conjugate.
[1034] The composition can include various materials that modify
the physical form of a solid or liquid dosage unit. For example,
the composition can include materials that form a coating shell
around the active ingredients. The materials that form the coating
shell are typically inert, and can be selected from, for example,
sugar, shellac, and other enteric coating agents. Alternatively,
the active ingredients can be encased in a gelatin capsule.
[1035] The compositions can consist of gaseous dosage units, e.g.,
it can be in the form of an aerosol. The term aerosol is used to
denote a variety of systems ranging from those of colloidal nature
to systems consisting of pressurized packages. Delivery can be by a
liquefied or compressed gas or by a suitable pump system that
dispenses the active ingredients.
[1036] Whether in solid, liquid or gaseous form, the present
compositions can include a pharmacological agent used in the
treatment of cancer, an autoimmune disease or an infectious
disease.
4.8 Therapeutic Uses of the Exemplary Conjugates
[1037] The Exemplary Compounds and/or Exemplary Conjugates are
useful for treating cancer, an autoimmune disease or an infectious
disease in a patient.
4.8.1 Treatment of Cancer
[1038] The Exemplary Compounds and/or Exemplary Conjugates are
useful for inhibiting the multiplication of a tumor cell or cancer
cell, causing apoptosis in a tumor or cancer cell, or for treating
cancer in a patient. The Exemplary Compounds and/or Exemplary
Conjugates can be used accordingly in a variety of settings for the
treatment of animal cancers. The Drug-Linker-Ligand Conjugates can
be used to deliver a Drug or Drug unit to a tumor cell or cancer
cell. Without being bound by theory, in one embodiment, the Ligand
unit of an Exemplary Conjugate binds to or associates with a
cancer-cell or a tumor-cell-associated antigen, and the Exemplary
Conjugate can be taken up inside a tumor cell or cancer cell
through receptor-mediated endocytosis. The antigen can be attached
to a tumor cell or cancer cell or can be an extracellular matrix
protein associated with the tumor cell or cancer cell. Once inside
the cell, one or more specific peptide sequences within the Linker
unit are hydrolytically cleaved by one or more tumor-cell or
cancer-cell-associated proteases, resulting in release of a Drug or
a Drug-Linker Compound. The released Drug or Drug-Linker Compound
is then free to migrate within the cell and induce cytotoxic or
cytostatic activities. In an alternative embodiment, the Drug or
Drug unit is cleaved from the Exemplary Conjugate outside the tumor
cell or cancer cell, and the Drug or Drug-Linker Compound
subsequently penetrates the cell.
[1039] In one embodiment, the Ligand unit binds to the tumor cell
or cancer cell.
[1040] In another embodiment, the Ligand unit binds to a tumor cell
or cancer cell antigen which is on the surface of the tumor cell or
cancer cell.
[1041] In another embodiment, the Ligand unit binds to a tumor cell
or cancer cell antigen which is an extracellular matrix protein
associated with the tumor cell or cancer cell.
[1042] The specificity of the Ligand unit for a particular tumor
cell or cancer cell can be important for determining those tumors
or cancers that are most effectively treated. For example,
Exemplary Conjugates having a BR96 Ligand unit can be useful for
treating antigen positive carcinomas including those of the lung,
breast, colon, ovaries, and pancreas. Exemplary Conjugates having
an Anti-CD30 or an anti-CD40 Ligand unit can be useful for treating
hematologic malignancies.
[1043] Other particular types of cancers that can be treated with
Exemplary Conjugates include, but are not limited to, those
disclosed in Table 3.
TABLE-US-00045 TABLE 3 Solid tumors, including but not limited to:
fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic
sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma
lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor
leiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer
kidney cancer pancreatic cancer bone cancer breast cancer ovarian
cancer prostate cancer esophogeal cancer stomach cancer oral cancer
nasal cancer throat cancer squamous cell carcinoma basal cell
carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland
carcinoma papillary carcinoma papillary adenocarcinomas
cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal
cell carcinoma hepatoma bile duct carcinoma choriocarcinoma
seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine
cancer testicular cancer small cell lung carcinoma bladder
carcinoma lung cancer epithelial carcinoma glioma glioblastoma
multiforme astrocytoma medulloblastoma craniopharyngioma ependymoma
pinealoma hemangioblastoma acoustic neuroma oligodendroglioma
meningioma skin cancer melanoma neuroblastoma retinoblastoma
blood-borne cancers, including but not limited to: acute
lymphoblastic leukemia "ALL" acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia acute myeloblastic leukemia
"AML" acute promyelocytic leukemia "APL" acute monoblastic leukemia
acute erythroleukemic leukemia acute megakaryoblastic leukemia
acute myelomonocytic leukemia acute nonlymphocyctic leukemia acute
undifferentiated leukemia chronic myelocytic leukemia "CML" chronic
lymphocytic leukemia "CLL" hairy cell leukemia multiple myeloma
acute and chronic leukemias: lymphoblastic myelogenous lymphocytic
myelocytic leukemias Lymphomas: Hodgkin's disease non-Hodgkin's
Lymphoma Multiple myeloma Waldenstrom's macroglobulinemia Heavy
chain disease Polycythemia vera
[1044] The Exemplary Conjugates provide conjugation-specific tumor
or cancer targeting, thus reducing general toxicity of these
compounds. The Linker units stabilize the Exemplary Conjugates in
blood, yet are cleavable by tumor-specific proteases within the
cell, liberating a Drug.
4.8.2 Multi-Modality Therapy for Cancer
[1045] Cancers, including, but not limited to, a tumor, metastasis,
or other disease or disorder characterized by uncontrolled cell
growth, can be treated or prevented by administration of an
Exemplary Conjugate and/or an Exemplary Compound.
[1046] In other embodiments, methods for treating or preventing
cancer are provided, including administering to a patient in need
thereof an effective amount of an Exemplary Conjugate and a
chemotherapeutic agent. In one embodiment the chemotherapeutic
agent is that with which treatment of the cancer has not been found
to be refractory. In another embodiment, the chemotherapeutic agent
is that with which the treatment of cancer has been found to be
refractory. The Exemplary Conjugates can be administered to a
patient that has also undergone surgery as treatment for the
cancer.
[1047] In one embodiment, the additional method of treatment is
radiation therapy.
[1048] In a specific embodiment, the Exemplary Conjugate is
administered concurrently with the chemotherapeutic agent or with
radiation therapy. In another specific embodiment, the
chemotherapeutic agent or radiation therapy is administered prior
or subsequent to administration of an Exemplary Conjugates, in one
aspect at least an hour, five hours, 12 hours, a day, a week, a
month, in further aspects several months (e.g., up to three
months), prior or subsequent to administration of an Exemplary
Conjugate.
[1049] A chemotherapeutic agent can be administered over a series
of sessions. Any one or a combination of the chemotherapeutic
agents listed in Table 4 can be administered. With respect to
radiation, any radiation therapy protocol can be used depending
upon the type of cancer to be treated. For example, but not by way
of limitation, x-ray radiation can be administered; in particular,
high-energy megavoltage (radiation of greater that 1 MeV energy)
can be used for deep tumors, and electron beam and orthovoltage
x-ray radiation can be used for skin cancers. Gamma-ray emitting
radioisotopes, such as radioactive isotopes of radium, cobalt and
other elements, can also be administered.
[1050] Additionally, methods of treatment of cancer with an
Exemplary Compound and/or Exemplary Conjugate are provided as an
alternative to chemotherapy or radiation therapy where the
chemotherapy or the radiation therapy has proven or can prove too
toxic, e.g., results in unacceptable or unbearable side effects,
for the subject being treated. The animal being treated can,
optionally, be treated with another cancer treatment such as
surgery, radiation therapy or chemotherapy, depending on which
treatment is found to be acceptable or bearable.
[1051] The Exemplary Compounds and/or Exemplary Conjugates can also
be used in an in vitro or ex vivo fashion, such as for the
treatment of certain cancers, including, but not limited to
leukemias and lymphomas, such treatment involving autologous stem
cell transplants. This can involve a multi-step process in which
the animal's autologous hematopoietic stem cells are harvested and
purged of all cancer cells, the animal's remaining bone-marrow cell
population is then eradicated via the administration of a high dose
of an Exemplary Compound and/or Exemplary Conjugate with or without
accompanying high dose radiation therapy, and the stem cell graft
is infused back into the animal. Supportive care is then provided
while bone marrow function is restored and the animal recovers.
4.8.3 Multi-Drug Therapy for Cancer
[1052] Methods for treating cancer including administering to a
patient in need thereof an effective amount of an Exemplary
Conjugate and another therapeutic agent that is an anti-cancer
agent are disclosed. Suitable anticancer agents include, but are
not limited to, methotrexate, taxol, L-asparaginase,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,
cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan,
camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,
vincristine, vinorelbine, paclitaxel, and docetaxel. In one aspect,
the anti-cancer agent includes, but is not limited to, a drug
listed in Table 4.
TABLE-US-00046 TABLE 4 Alkylating agents Nitrogen mustards:
cyclophosphamide ifosfamide trofosfamide chlorambucil melphalan
Nitrosoureas: carmustine (BCNU) lomustine (CCNU) Alkylsulphonates
busulfan treosulfan Triazenes: decarbazine Platinum containing
compounds: cisplatin carboplatin Plant Alkaloids Vinca alkaloids:
vincristine vinblastine vindesine vinorelbine Taxoids: paclitaxel
docetaxol DNA Topoisomerase Inhibitors Epipodophyllins: etoposide
teniposide topotecan 9-aminocamptothecin camptothecin crisnatol
mitomycins: mitomycin C Anti-metabolites Anti-folates: DHFR
inhibitors: methotrexate trimetrexate IMP dehydrogenase Inhibitors:
mycophenolic acid tiazofurin ribavirin EICAR Ribonucleotide
reductase Inhibitors: hydroxyurea deferoxamine Pyrimidine analogs:
Uracil analogs 5-Fluorouracil floxuridine doxifluridine ratitrexed
Cytosine analogs cytarabine (ara C) cytosine arabinoside
fludarabine Purine analogs: mercaptopurine thioguanine Hormonal
therapies: Receptor antagonists: Anti-estrogen tamoxifen raloxifene
megestrol LHRH agonists: goscrclin leuprolide acetate
Anti-androgens: flutamide bicalutamide Retinoids/Deltoids Vitamin
D3 analogs: EB 1089 CB 1093 KH 1060 Photodynamic therapies:
vertoporfin (BPD-MA) phthalocyanine photosensitizer Pc4
demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines: Interferon-.alpha.
Interferon-.gamma. tumor necrosis factor Others: Gemcitabine
Velcade Revamid Thalamid Isoprenylation inhibitors: Lovastatin
Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell
cycle inhibitors: staurosporine Actinomycins: Actinomycin D
dactinomycin Bleomycins: bleomycin A2 bleomycin B2 peplomycin
Anthracyclines: daunorubicin Doxorubicin (adriamycin) idarubicin
epirubicin pirarubicin zorubicin mtoxantrone MDR inhibitors:
verapamil Ca.sup.2+ATPase inhibitors: thapsigargin
4.8.4 Treatment of Autoimmune Diseases
[1053] The Exemplary Conjugates are useful for killing or
inhibiting the replication of a cell that produces an autoimmune
disease or for treating an autoimmune disease. The Exemplary
Conjugates can be used accordingly in a variety of settings for the
treatment of an autoimmune disease in a patient. The
Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a
target cell. Without being bound by theory, in one embodiment, the
Drug-Linker-Ligand Conjugate associates with an antigen on the
surface of a target cell, and the Exemplary Conjugate is then taken
up inside a target-cell through receptor-mediated endocytosis. Once
inside the cell, one or more specific peptide sequences within the
Linker unit are enzymatically or hydrolytically cleaved, resulting
in release of a Drug. The released Drug is then free to migrate in
the cytosol and induce cytotoxic or cytostatic activities. In an
alternative embodiment, the Drug is cleaved from the Exemplary
Conjugate outside the target cell, and the Drug subsequently
penetrates the cell.
[1054] In one embodiment, the Ligand unit binds to an autoimmune
antigen. In one aspect, the antigen is on the surface of a cell
involved in an autoimmune condition.
[1055] In another embodiment, the Ligand unit binds to an
autoimmune antigen which is on the surface of a cell.
[1056] In one embodiment, the Ligand binds to activated lymphocytes
that are associated with the autoimmune disease state.
[1057] In a further embodiment, the Exemplary Conjugates kill or
inhibit the multiplication of cells that produce an autoimmune
antibody associated with a particular autoimmune disease.
[1058] Particular types of autoimmune diseases that can be treated
with the Exemplary Conjugates include, but are not limited to, Th2
lymphocyte related disorders (e.g., atopic dermatitis, atopic
asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,
systemic sclerosis, and graft versus host disease); Th1
lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple
sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,
Grave's disease, primary biliary cirrhosis, Wegener's
granulomatosis, and tuberculosis); activated B lymphocyte-related
disorders (e.g., systemic lupus erythematosus, Goodpasture's
syndrome, rheumatoid arthritis, and type I diabetes); and those
disclosed in Table 5.
TABLE-US-00047 TABLE 5 Active Chronic Hepatitis Addison's Disease
Allergic Alveolitis Allergic Reaction Allergic Rhinitis Alport's
Syndrome Anaphlaxis Ankylosing Spondylitis Anti-phosholipid
Syndrome Arthritis Ascariasis Aspergillosis Atopic Allergy Atropic
Dermatitis Atropic Rhinitis Behcet's Disease Bird-Fancier's Lung
Bronchial Asthma Caplan's Syndrome Cardiomyopathy Celiac Disease
Chagas' Disease Chronic Glomerulonephritis Cogan's Syndrome Cold
Agglutinin Disease Congenital Rubella Infection CREST Syndrome
Crohn's Disease Cryoglobulinemia Cushing's Syndrome Dermatomyositis
Discoid Lupus Dressler's Syndrome Eaton-Lambert Syndrome Echovirus
Infection Encephalomyelitis Endocrine opthalmopathy Epstein-Barr
Virus Infection Equine Heaves Erythematosis Evan's Syndrome Felty's
Syndrome Fibromyalgia Fuch's Cyclitis Gastric Atrophy
Gastrointestinal Allergy Giant Cell Arteritis Glomerulonephritis
Goodpasture's Syndrome Graft v. Host Disease Graves' Disease
Guillain-Barre Disease Hashimoto's Thyroiditis Hemolytic Anemia
Henoch-Schonlein Purpura Idiopathic Adrenal Atrophy Idiopathic
Pulmonary Fibritis IgA Nephropathy Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus Juvenile Arthritis Juvenile
Diabetes Mellitus (Type I) Lambert-Eaton Syndrome Laminitis Lichen
Planus Lupoid Hepatitis Lupus Lymphopenia Meniere's Disease Mixed
Connective Tissue Disease Multiple Sclerosis Myasthenia Gravis
Pernicious Anemia Polyglandular Syndromes Presenile Dementia
Primary Agammaglobulinemia Primary Biliary Cirrhosis Psoriasis
Psoriatic Arthritis Raynauds Phenomenon Recurrent Abortion Reiter's
Syndrome Rheumatic Fever Rheumatoid Arthritis Sampter's Syndrome
Schistosomiasis Schmidt's Syndrome Scleroderma Shulman's Syndrome
Sjorgen's Syndrome Stiff-Man Syndrome Sympathetic Ophthalmia
Systemic Lupus Erythematosis Takayasu's Arteritis Temporal
Arteritis Thyroiditis Thrombocytopenia Thyrotoxicosis Toxic
Epidermal Necrolysis Type B Insulin Resistance Type I Diabetes
Mellitus Ulcerative Colitis Uveitis Vitiligo Waldenstrom's
Macroglobulemia Wegener's Granulomatosis
4.8.5 Multi-Drug Therapy of Autoimmune Diseases
[1059] Methods for treating an autoimmune disease are also
disclosed including administering to a patient in need thereof an
effective amount of an Exemplary Conjugate and another therapeutic
agent known for the treatment of an autoimmune disease. In one
embodiment, the anti-autoimmune disease agent includes, but is not
limited to, agents listed in Table 6.
TABLE-US-00048 TABLE 6 cyclosporine cyclosporine A mycophenylate
mofetil sirolimus tacrolimus enanercept prednisone azathioprine
methotrexate cyclophosphamide prednisone aminocaproic acid
chloroquine hydroxychloroquine hydrocortisone dexamethasone
chlorambucil DHEA danazol bromocriptine meloxicam infliximab
4.8.6 Treatment of Infectious Diseases
[1060] The Exemplary Conjugates are useful for killing or
inhibiting the multiplication of a cell that produces an infectious
disease or for treating an infectious disease. The Exemplary
Conjugates can be used accordingly in a variety of settings for the
treatment of an infectious disease in a patient. The
Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a
target cell. In one embodiment, the Ligand unit binds to the
infectious disease cell.
[1061] In one embodiment, the Conjugates kill or inhibit the
multiplication of cells that produce a particular infectious
disease.
[1062] Particular types of infectious diseases that can be treated
with the Exemplary Conjugates include, but are not limited to,
those disclosed in Table 7.
TABLE-US-00049 TABLE 7 Bacterial Diseases: Diphtheria Pertussis
Occult Bacteremia Urinary Tract Infection Gastroenteritis
Cellulitis Epiglottitis Tracheitis Adenoid Hypertrophy
Retropharyngeal Abcess Impetigo Ecthyma Pneumonia Endocarditis
Septic Arthritis Pneumococcal Peritonitis Bactermia Meningitis
Acute Purulent Meningitis Urethritis Cervicitis Proctitis
Pharyngitis Salpingitis Epididymitis Gonorrhea Syphilis Listeriosis
Anthrax Nocardiosis Salmonella Typhoid Fever Dysentery
Conjunctivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic
Plague Tetanus Necrotizing Enteritis Actinomycosis Mixed Anaerobic
Infections Syphilis Relapsing Fever Leptospirosis Lyme Disease Rat
Bite Fever Tuberculosis Lymphadenitis Leprosy Chlamydia Chlamydial
Pneumonia Trachoma Inclusion Conjunctivitis Systemic Fungal
Diseases: Histoplamosis Coccidiodomycosis Blastomycosis
Sporotrichosis Cryptococcsis Systemic Candidiasis Aspergillosis
Mucormycosis Mycetoma Chromomycosis Rickettsial Diseases: Typhus
Rocky Mountain Spotted Fever Ehrlichiosis Eastern Tick-Borne
Rickettsioses Rickettsialpox Q Fever Bartonellosis Parasitic
Diseases: Malaria Babesiosis African Sleeping Sickness Chagas'
Disease Leishmaniasis Dum-Dum Fever Toxoplasmosis
Meningoencephalitis Keratitis Entamebiasis Giardiasis
Cryptosporidiasis Isosporiasis Cyclosporiasis Microsporidiosis
Ascariasis Whipworm Infection Hookworm Infection Threadworm
Infection Ocular Larva Migrans Trichinosis Guinea Worm Disease
Lymphatic Filariasis Loiasis River Blindness Canine Heartworm
Infection Schistosomiasis Swimmer's Itch Oriental Lung Fluke
Oriental Liver Fluke Fascioliasis Fasciolopsiasis Opisthorchiasis
Tapeworm Infections Hydatid Disease Alveolar Hydatid Disease Viral
Diseases: Measles Subacute sclerosing panencephalitis Common Cold
Mumps Rubella Roseola Fifth Disease Chickenpox Respiratory
syncytial virus infection Croup Bronchiolitis Infectious
Mononucleosis Poliomyelitis Herpangina Hand-Foot-and-Mouth Disease
Bornholm Disease Genital Herpes Genital Warts Aseptic Meningitis
Myocarditis Pericarditis Gastroenteritis Acquired Immunodeficiency
Syndrome (AIDS) Human Immunodeficiency Virus (HIV) Reye's Syndrome
Kawasaki Syndrome Influenza Bronchitis Viral "Walking" Pneumonia
Acute Febrile Respiratory Disease Acute pharyngoconjunctival fever
Epidemic keratoconjunctivitis Herpes Simplex Virus 1 (HSV-1) Herpes
Simplex Virus 2 (HSV-2) Shingles Cytomegalic Inclusion Disease
Rabies Progressive Multifocal Leukoencephalopathy Kuru Fatal
Familial Insomnia Creutzfeldt-Jakob Disease
Gerstmann-Straussler-Scheinker Disease Tropical Spastic Paraparesis
Western Equine Encephalitis California Encephalitis St. Louis
Encephalitis Yellow Fever Dengue Lymphocytic choriomeningitis Lassa
Fever Hemorrhagic Fever Hantvirus Pulmonary Syndrome Marburg Virus
Infections Ebola Virus Infections Smallpox
4.8.7 Multi-Drug Therapy of Infectious Diseases
[1063] Methods for treating an infectious disease are disclosed
including administering to a patient in need thereof an Exemplary
Conjugate and another therapeutic agent that is an anti-infectious
disease agent. In one embodiment, the anti-infectious disease agent
is, but not limited to, agents listed in Table 8.
TABLE-US-00050 TABLE 8 .beta.-Lactam Antibiotics: Penicillin G
Penicillin V Cloxacilliin Dicloxacillin Methicillin Nafcillin
Oxacillin Ampicillin Amoxicillin Bacampicillin Azlocillin
Carbenicillin Mezlocillin Piperacillin Ticarcillin Aminoglycosides:
Amikacin Gentamicin Kanamycin Neomycin Netilmicin Streptomycin
Tobramycin Macrolides: Azithromycin Clarithromycin Erythromycin
Lincomycin Clindamycin Tetracyclines: Demeclocycline Doxycycline
Minocycline Oxytetracycline Tetracycline Quinolones: Cinoxacin
Nalidixic Acid Fluoroquinolones: Ciprofloxacin Enoxacin
Grepafloxacin Levofloxacin Lomefloxacin Norfloxacin Ofloxacin
Sparfloxacin Trovafloxicin Polypeptides: Bacitracin Colistin
Polymyxin B Sulfonamides: Sulfisoxazole Sulfamethoxazole
Sulfadiazine Sulfamethizole Sulfacetamide Miscellaneous
Antibacterial Agents: Trimethoprim Sulfamethazole Chloramphenicol
Vancomycin Metronidazole Quinupristin Dalfopristin Rifampin
Spectinomycin Nitrofurantoin Antiviral Agents: General Antiviral
Agents: Idoxuradine Vidarabine Trifluridine Acyclovir Famcicyclovir
Pencicyclovir Valacyclovir Gancicyclovir Foscarnet Ribavirin
Amantadine Rimantadine Cidofovir Antisense Oligonucleotides
Immunoglobulins Inteferons Drugs for HIV infection: Tenofovir
Emtricitabine Zidovudine Didanosine Zalcitabine Stavudine
Lamivudine Nevirapine Delavirdine Saquinavir Ritonavir Indinavir
Nelfinavir
5. EXAMPLES
Example 1
Preparation of Compound Ab
##STR00070##
[1065] Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat.
No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL,
0.2 M) and to this solution was added a diethylamine (60 mL). The
reaction was monitored by HPLC and found to be complete in 2 h. The
reaction mixture was concentrated and the resulting residue was
precipitated using ethyl acetate (ca. 100 mL) under sonication over
for 10 min. Ether (200 mL) was added and the precipitate was
further sonicated for 5 min. The solution was allowed to stand for
30 min. without stirring and was then filtered and dried under high
vacuum to provide Val-cit-PAB-OH, which was used in the next step
without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH
(8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting
solution was treated with MC-OSu (Willner et al., (1993)
Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was
complete according to HPLC after 2 h. The reaction mixture was
concentrated and the resulting oil was precipitated using ethyl
acetate (50 mL). After sonicating for 15 min, ether (400 mL) was
added and the mixture was sonicated further until all large
particles were broken up. The solution was then filtered and the
solid dried to provide an off-white solid intermediate. Yield:
11.63 g (96%); ES-MS m/z 757.9 [M-H]
[1066] Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat.
No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL,
0.2 M) and to this solution was added a diethylamine (60 mL). The
reaction was monitored by HPLC and found to be complete in 2 h. The
reaction mixture was concentrated and the resulting residue was
precipitated using ethyl acetate (ca. 100 mL) under sonication over
for 10 min. Ether (200 mL) was added and the precipitate was
further sonicated for 5 min. The solution was allowed to stand for
30 min. without stirring and was then filtered and dried under high
vacuum to provide Val-cit-PAB-OH, which was used in the next step
without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH
(8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting
solution was treated with MC-OSu (Willner et al., (1993)
Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was
complete according to HPLC after 2 h. The reaction mixture was
concentrated and the resulting oil was precipitated using ethyl
acetate (50 mL). After sonicating for 15 min, ether (400 mL) was
added and the mixture was sonicated further until all large
particles were broken up. The solution was then filtered and the
solid dried to provide an off-white solid intermediate. Yield:
11.63 g (96%); ES-MS m/z 757.9 [M-H].
[1067] The off-white solid intermediate (8.0 g, 14.0 mmol) was
diluted with DMF (120 mL, 0.12 M) and to the resulting solution was
added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and
DIEA (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1
h according to HPLC. The reaction mixture was concentrated to
provide an oil that was precipitated with EtOAc, and then
triturated with EtOAc (ca. 25 mL). The solute was further
precipitated with ether (ca. 200 mL) and triturated for 15 min. The
solid was filtered and dried under high vacuum to provide Compound
AB which was 93% pure according to HPLC and used in the next step
without further purification. Yield: 9.7 g (94%).
Example 2
Preparation of Compound 1
##STR00071##
[1069] Phenylalanine t-butyl ester HCl salt (868 mg, 3 mmol),
N-Boc-Dolaproine (668 mg, 1 eq.), DEPC (820 .mu.L, 1.5 eq.), and
DIEA (1.2 mL) were diluted with dichloromethane (3 mL). After 2
hours (h) at room temperature (about 28 degrees Celsius), the
reaction mixture was diluted with dichloromethane (20 mL), washed
successively with saturated aqueous (aq.) NaHCO.sub.3 (2.times.10
mL), saturated aq. NaCl (2.times.10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography in ethyl
acetate. The relevant fractions were combined and concentrated to
provide the dipeptide as a white solid: 684 mg (46%). ES-MS m/z
491.3 [M+H].sup.+.
[1070] For selective Boc cleavage in the presence of t-butyl ester,
the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2
mL). 4M HCl/dioxane (960 .mu.L, 3 eq.) was added, and the reaction
mixture was stirred overnight at room temperature. Almost complete
Boc deprotection was observed by RP-HPLC with minimal amount of
t-butyl ester cleavage. The mixture was cooled down on an ice bath,
and triethylamine (500 .mu.L) was added. After 10 min., the mixture
was removed from the cooling bath, diluted with dichloromethane (20
mL), washed successively with saturated aq. NaHCO.sub.3 (2.times.10
mL), saturated aq. NaCl (2.times.10 mL). The organic layer was
concentrated to give a yellow foam: 287 mg (57%). The intermediate
was used without further purification.
[1071] The tripeptide Fmoc-Meval-val-dil-O-t-Bu (prepared as
described in WO 02/088172, entitled "Pentapeptide Compounds and
Uses Related Thereto"; 0.73 mmol) was treated with TFA (3 mL),
dichloromethane (3 mL) for 2 h at room temperature. The mixture was
concentrated to dryness, the residue was co-evaporated with toluene
(3.times.20 mL), and dried in vacuum overnight. The residue was
diluted with dichloromethane (5 mL) and added to the deprotected
dipeptide (287 mg, 0.73 mmol), followed by DIEA (550 .mu.L, 4 eq.),
DEPC (201 .mu.L, 1.1 eq.). After 2 h at room temperature the
reaction mixture was diluted with ethyl acetate (50 mL), washed
successively with 10% aq. citric acid (2.times.20 mL), saturated
aq. NaHCO.sub.3 (2.times.10 mL), saturated aq. NaCl (10 mL). The
organic layer was separated and concentrated. The resulting residue
was re-suspended in ethyl acetate and was purified via flash
chromatography in ethyl acetate. The relevant fractions were
combined and concentrated to provide
Fmoc-Meval-val-dil-dap-phe-O-t-Bu as a white solid: 533 mg (71%).
R.sub.f 0.4 (EtOAc). ES-MS m/z 1010.6 [M+H].sup.+.
[1072] The product (200 mg, 0.2 mmol) was diluted with
dichloromethane (3 mL), diethylamine (1 mL). The reaction mixture
was stirred overnight at room temperature. Solvents were removed to
provide an oil that was purified by flash silica gel chromatography
in a step gradient 0-10% MeOH in dichloromethane to provide
Compound 1 as a white solid: 137 mg (87%). R.sub.f 0.3 (10%
MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 788.6 [M+H].sup.+.
Example 3
Preparation of Compound 2
##STR00072##
[1074] Compound 2 was prepared from compound 1 (30 mg, 0.038 mmol)
by treatment with 4M HCl/dioxane (4 ml) for 7 h at room
temperature. The solvent was removed, and the residue was dried in
a vacuum overnight to give provide Compound 2 as a hydroscopic
white solid: 35 mg (120% calculated for HCl salt). ES-MS m/z 732.56
[M+H].sup.+.
Example 4
Preparation of Compound 3
##STR00073##
[1076] Fmoc-Meval-val-dil-dap-phe-O-t-Bu (Example 2, 50 mg) was
treated with 4M HCl/dioxane (4 ml) for 16 h at room temperature.
The solvent was removed, and the residue was dried in vacuum
overnight to give 50 mg of a hydroscopic white solid intermediate
The white solid intermediate (20 mg, 0.02 mmol) was diluted with
dichloromethane (1 mL); DEPC (5 .mu.L, 0.03 mmol, 1.5 eq.) was
added followed by DIEA (11 .mu.L, 0.06 mmol, 3 eq.), and
t-butylamine (3.2 .mu.L, 0.03 mmol, 1.5 eq.). After 2 h at room
temperature, the reaction was found to be uncompleted by RP-HPLC.
More DEPC (10 .mu.L) and t-butylamine (5 .mu.L) were added and the
reaction was stirred for additional 4 h. Reaction mixture was
diluted with dichloromethane (15 mL), washed successively with
water (5 mL), 0.1 M aq. HCl (10 mL), saturated aq. NaCl (10 mL).
The organic layer was separated and concentrated. The resulting
residue was diluted with dichloromethane and purified via flash
chromatography in a step gradient 0-5% MeOH in dichloromethane. The
relevant fractions were combined and concentrated to provide the
Fmoc protected intermediate as a white solid: 7.3 mg (36%). R.sub.f
0.75 (10% MeOH/CH.sub.2Cl.sub.2).
[1077] Fmoc protected intermediate was diluted with dichloromethane
(0.5 mL) and treated with diethylamine (0.5 mL) for 3 h at room
temperature. The reaction mixture was concentrated to dryness. The
product was isolated by flash silica gel chromatography in a step
gradient 0-10% MeOH in dichloromethane to provide Compound 3 as a
white solid: 4 mg (70%). R.sub.f 0.2 (10% MeOH/CH.sub.2Cl.sub.2).
ES-MS m/z 787 [M+H].sup.+, 809 [M+Na].sup.+.
Example 5
Preparation of Compound 4
##STR00074##
[1079] Boc-L-Phenylalanine (265 mg, 1 mmol, 1 eq.) and
triethyleneglycol monomethyl ether (164 .mu.L, 1 mmol, 1 eq.) were
diluted with dichloromethane (5 mL). Then, DCC (412 mg, 2 mmol, 2
eq.) was added, followed by DMAP (10 mg). The reaction mixture was
stirred overnight at room temperature. The precipitate was filtered
off. The solvent was removed in a vacuum, the residue was diluted
with ethyl acetate, and purified by silica gel flash chromatography
in ethyl acetate. The product containing fractions were pulled,
concentrated, and dried in vacuum to give a white solid: 377 mg
(91%). R.sub.f 0.5 (EtOAc). ES-MS m/z 434 [M+Na].sup.+.
[1080] Removal of Boc protecting group was performed by treatment
of the above material in dioxane (10 mL) with 4M HCl/dioxane (6 mL)
for 6 h at room temperature. The solvent was removed in a vacuum,
the residue was dried in a vacuum to give a white solid.
[1081] The HCl salt of Phenylalanine-triethyleneglycol monomethyl
ether ester (236 mg, 0.458 mmol, 1 eq.) and N-Boc-Dolaproine (158
mg, 0.55 mmol, 1.2 eq.) were diluted with dichloromethane (3 mL).
DEPC (125 .mu.L, 1.5 eq.) and added to the mixture followed by DIEA
(250 .mu.L, 3 eq.). After 2 h at room temperature the reaction
mixture was diluted with ethyl acetate (30 mL), washed successively
with saturated aq. NaHCO.sub.3 (2.times.10 mL), 10% aq. citric acid
(2.times.10 mL), saturated aq. NaCl (10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography on
silica gel in ethyl acetate. The relevant fractions were combined
and concentrated to provide a white foam intermediate: 131 mg
(50%). R.sub.f 0.25 (EtOAc). ES-MS m/z 581.3 [M+H].sup.+.
[1082] Boc deprotection was done in dichloromethane (2 mL), TFA
(0.5 mL) at room temperature for 2 h. Solvent was removed in
vacuum, and the residue was co-evaporated with toluene (3.times.25
mL), then dried in vacuum to give 138 mg of dipeptide TFA salt.
[1083] Fmoc-Meval-val-dil-OH (Example 2, 147 mg, 0.23 mmol, 1 eq.),
and dipeptide TFA salt (138 mg) were diluted with dichloromethane
(2 mL). To the mixture DEPC (63 .mu.L, 1.5 eq.) was added, followed
by DIEA (160 .mu.L, 4 eq.). After 2 h at room temperature the
reaction mixture was diluted with dichloromethane (30 mL), washed
successively with 10% aq. citric acid (2.times.20 mL), saturated
aq. NaCl (20 mL). The organic layer was separated and concentrated.
The resulting residue was re-suspended in dichloromethane and was
purified via flash chromatography on silica gel in a step gradient
0-5% MeOH in dichloromethane. The relevant fractions were combined
and concentrated to provide white foam: 205 mg (81%). R.sub.f 0.4
(10% MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 1100.6 [M+H].sup.+, 1122.4
[M+Na].sup.+.
[1084] Fmoc protecting group was removed by treatment with
diethylamine (2 mL) in dichloromethane (6 mL). After 6 h at room
temperature solvent was removed in vacuum, product was isolated by
flash chromatography on silica gel in a step gradient 0-10% MeOH in
dichloromethane. The relevant fractions were combined and
concentrated. After evaporation from dichloromethane/hexane, 1:1,
Compound 4 was obtained as a white foam: 133 mg (80%). R.sub.f 0.15
(10% MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 878.6 [M+H].sup.+.
Example 6
Preparation of Compound 5
##STR00075##
[1086] Fmoc-Meval-val-dil-OH (Example 2, 0.50 g, 0.78 mmol) and
dap-phe-Ome.HCl (0.3 g, 0.78 mmol, prepared according to Pettit, G.
R., et al. Anti-Cancer Drug Design 1998, 13, 243-277) were
dissolved in CH.sub.2Cl.sub.2 (10 mL) followed by the addition of
diisopropylethylamine (0.30 mL, 1.71 mmol, 2.2 eq.). DEPC (0.20 mL,
1.17, 1.5 eq.) was added and the contents stood over Ar. Reaction
was complete according to HPLC in 1 h. The mixture was concentrated
to an oil and purified by SiO.sub.2 chromatography (300.times.25 mm
column) and eluting with 100% EtOAc. The product was isolated as a
white foamy solid. Yield: 0.65 g (87%). ES-MS m/z 968.35
[M+H].sup.+, 991.34 [M+Na].sup.+; UV .lamda..sub.max 215, 265
nm.
[1087] The Fmoc-protected peptide (0.14 g, 0.14 mmol) in methylene
chloride (5 mL) was treated with diethylamine (2 mL) and the
contents stood at room temperature for 2 h. The reaction, complete
by HPLC, was concentrated to an oil, taken up in 2 mL of DMSO and
injected into a preparative-HPLC(C.sub.12-RP column, 5.mu., 100
.ANG., linear gradient of MeCN in water (containing 0.1% TFA) 10 to
100% in 40 min followed by 20 min at 100%, at a flow rate of 25
mL/min). Fractions containing the product were evaporated to afford
a white powder for the trifluoroacetate salt. Yield: 0.126 g (98%).
R.sub.f 0.28 (100% EtOAc); ES-MS m/z 746.59 [M+H].sup.+, 768.51
[M+Na].sup.+; UV .lamda..sub.max 215 nm.
Example 7
Preparation of Compound 6
##STR00076##
[1089] The trifluoroacetate salt of Compound 5 (0.11 g, 0.13 mmol),
Compound AB (0.103 g, 0.14 mmol, 1.1 eq.) and HOBt (3.4 mg, 26
.mu.mol, 0.2 eq.) were suspended in DMF/pyridine (2 mL/0.5 mL,
respectively). Diisopropylethylamine (22.5 .mu.t, 0.13 mmol, 1.0
eq.) was added and the yellow solution stirred while under argon.
After 3 h, an additional 1.0 eq. of DIEA was added. 24 hours later,
0.5 eq. of the activated linker was included in the reaction
mixture. After 40 h total, the reaction was complete. The contents
were evaporated, taken up in DMSO and injected into a
prep-HPLC(C.sub.12-RP column, 5.mu., 100 .ANG., linear gradient of
MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed
by 20 min at 100%, at a flow rate of 50 mL/min). The desired
fractions were evaporated to give the product as a yellow oil.
Methylene chloride (ca. 2 mL) and excess ether were added to
provide Compound 6 as a white precipitate that was filtered and
dried. Yield: 90 mg (52%). ES-MS m/z 1344.32 [M+H].sup.+, 1366.29
[M+Na].sup.+; UV .lamda..sub.max 215, 248 nm.
Example 8
Preparation of Compound 7
##STR00077##
[1091] Compound 4 (133 mg, 0.15 mmol, 1 eq.), Compound AB, (123 mg,
0.167 mmol, 1.1 eq.), and HOBt (4 mg, 0.2 eq.) were diluted with
DMF (1.5 mL). After 2 min, pyridine (5 mL) was added and the
reaction was monitored using RP-HPLC. The reaction was shown to be
complete within 18 h. The reaction mixture was diluted with
dichloromethane (20 mL), washed successively with 10% aq. citric
acid (2.times.10 mL), water (10 mL), saturated aq. NaCl (10 mL).
The organic layer was separated and concentrated. The resulting
residue was re-suspended in dichloromethane and was purified via
flash chromatography on silica gel in a step gradient 0-10% MeOH in
dichloromethane. The relevant fractions were combined and
concentrated to provide Compound 7 as a white foam: 46 mg (21%).
R.sub.f 0.15 (10% MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 1476.94
[M+H].sup.+.
Example 9
Preparation of MC-Val-Cit-PAB-MMAF t-butyl ester 8
##STR00078##
[1093] Compound 1 (83 mg, 0.11 mmol), Compound AB (85 mg, 0.12
mmol, 1.1 eq.), and HOBt (2.8 mg, 21 .mu.mol, 0.2 eq.) were taken
up in dry DMF (1.5 mL) and pyridine (0.3 mL) while under argon.
After 30 h, the reaction was found to be essentially complete by
HPLC. The mixture was evaporated, taken up in a minimal amount of
DMSO and purified by prep-HPLC(C.sub.12-RP column, 5.mu., 100
.ANG., linear gradient of MeCN in water (containing 0.1% TFA) 10 to
100% in 40 min followed by 20 min at 100%, at a flow rate of 25
mL/min) to provide Compound 8 as a white solid. Yield: 103 mg
(71%). ES-MS m/z 1387.06 [M+H].sup.+, 1409.04 [M+Na].sup.+; UV
.lamda..sub.max 205, 248 nm.
Example 10
Preparation of MC-val-cit-PAB-MMAF 9
##STR00079##
[1095] Compound 8 (45 mg, 32 .mu.mol) was suspended in methylene
chloride (6 mL) followed by the addition of TFA (3 mL). The
resulting solution stood for 2 h. The reaction mixture was
concentrated in vacuo and purified by prep-HPLC (C.sub.12-RP
column, 5.mu., 100 .ANG., linear gradient of MeCN in water
(containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at
100%, at a flow rate of 25 mL/min). The desired fractions were
concentrated to provide
maleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF
(MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%).
ES-MS m/z 1330.29 [M+H].sup.+, 1352.24 [M+Na].sup.+; UV
.lamda..sub.max 205, 248 nm.
Example 11
Preparation of MC-val-cit-PAB-MMAF tert-butyl amide 10
##STR00080##
[1097] Compound 3 (217 mg, 0.276 mmol, 1.0 eq.), Compound AB (204
mg, 0.276 mmol, 1.0 eq.), and HOBt (11 mg, 0.0828 mmol, 0.3 eq.)
were diluted with pyridine/DMF (6 mL). To this mixture was added
DIEA (0.048 mL), and the mixture was stirred ca. 16 hr. Volatile
organics were evaporated in vacuo. The crude residue was purified
by Chromatotron.RTM. (radial thin-layer chromatography) with a step
gradient (0-5-10% methanol in DCM) to provide MC-val-cit-PAB-MMAF
tert-butyl amide 10. Yield: 172 mg (45%); ES-MS m/z 1386.33
[M+H].sup.+, 1408.36 [M+Na].sup.+; UV .lamda..sub.max 215, 248
nm.
Example 12
Preparation of AC10-MC-MMAE by conjugation of AC10 and MC-MMAE
[1098] AC10, dissolved in 500 mM sodium borate and 500 mM sodium
chloride at pH 8.0 is treated with an excess of 100 mM
dithiothreitol (DTT). After incubation at 37.degree. C. for about
30 minutes, the buffer is exchanged by elution over Sephadex G25
resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is
checked by determining the reduced antibody concentration from the
absorbance at 280 nm of the solution and the thiol concentration by
reaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of
the absorbance at 412 nm. The reduced antibody dissolved in PBS is
chilled on ice.
[1099] The drug linker reagent, maleimidocaproyl-monomethyl
auristatin E, i.e. MC-MMAE, dissolved in DMSO, is diluted in
acetonitrile and water at known concentration, and added to the
chilled reduced antibody AC10 in PBS. After about one hour, an
excess of maleimide is added to quench the reaction and cap any
unreacted antibody thiol groups. The reaction mixture is
concentrated by centrifugal ultrafiltration and AC10-MC-MMAE is
purified and desalted by elution through G25 resin in PBS, filtered
through 0.2 .mu.m filters under sterile conditions, and frozen for
storage.
Example 13
Preparation of AC10-MC-MMAF by conjugation of AC10 and MC-MMAF
[1100] AC10-MC-MMAF was prepared by conjugation of AC10 and MC-MMAF
following the procedure of Example 12.
Example 14
Preparation of AC10-MC-val-cit-PAB-MMAE by conjugation of AC10 and
MC-val-cit-PAB-MMAE
[1101] AC10-MC-val-cit-PAB-MMAE was prepared by conjugation of AC10
and MC-val-cit-PAB-MMAE following the procedure of Example 12.
Example 15
Preparation of AC10-MC-val-cit-PAB-MMAF by conjugation of AC10 and
MC-val-cit-PAB-MMAF (9)
[1102] AC10-MC-val-cit-PAB-MMAF was prepared by conjugation of AC10
and MC-val-cit-PAB-MMAF (9) following the procedure of Example
12.
Example 16
Determination of Cytotoxicity of Selected Compounds
[1103] Cytotoxic activity of MMAF and Compounds 1-5 was evaluated
on the Lewis Y positive cell lines OVCAR-3, H3396 breast carcinoma,
L2987 lung carcinoma and LS174t colon carcinoma Lewis Y positive
cell lines can be assayed for cytotoxicity. To evaluate the
cytotoxicity of Compounds 1-5, cells can be seeded at approximately
5-10,000 per well in 150 .mu.l of culture medium then treated with
graded doses of Compounds 1-5 in quadruplicates at the initiation
of assay. Cytotoxicity assays are usually carried out for 96 hours
after addition of test compounds. Fifty .mu.l of resazurin dye may
be added to each well during the last 4 to 6 hours of the
incubation to assess viable cells at the end of culture. Dye
reduction can be determined by fluorescence spectrometry using the
excitation and emission wavelengths of 535 nm and 590 nm,
respectively. For analysis, the extent of resazurin reduction by
the treated cells can be compared to that of the untreated control
cells.
[1104] For 1 h exposure assays cells can be pulsed with the drug
for 1 h and then washed; the cytotoxic effect can be determined
after 96 h of incubation.
Example 17
In Vitro Cytotoxicity Cata for Selected Compounds
[1105] Table 10 shows cytotoxic effect of cAC10 Conjugates of
Compounds 7-10, assayed as described in General Procedure I on a
CD30+cell line Karpas 299. Data of two separate experiments are
presented. The cAC10 conjugates of Compounds 7 and 9 were found to
be slightly more active than cAC10-val-cit-MMAE.
TABLE-US-00051 TABLE 10 Conjugate IC.sub.50 (ng/mL)
cAC10-val-cit-MMAE 6 cAC10-7 1.0 cAC10-8 15 cAC10-9 0.5 cAC10-10
20
[1106] In other experiments, BR96-val-cit-MMAF was at least 250
fold more potent than the free MMAF.
[1107] General Procedure I--Cytotoxicity determination. To evaluate
the cytotoxicity of Exemplary Conjugates 7-10, cells were seeded at
approximately 5-10,000 per well in 150 d of culture medium then
treated with graded doses of Exemplary Conjugates 7-10 in
quadruplicates at the initiation of assay. Cytotoxicity assays were
carried out for 96 hours after addition of test compounds. Fifty
.mu.l of the resazurin dye was added to each well during the last 4
to 6 hours of the incubation to assess viable cells at the end of
culture. Dye reduction was determined by fluorescence spectrometry
using the excitation and emission wavelengths of 535 nm and 590 nm,
respectively. For analysis, the extent of resazurin reduction by
the treated cells was compared to that of the untreated control
cells.
Example 18
In Vitro Cell Proliferation Assay
[1108] Efficacy of ADC can be measured by a cell proliferation
assay employing the following protocol (Promega Corp. Technical
Bulletin TB288; Mendoza et al. (2002) Cancer Res.
62:5485-5488):
1. An aliquot of 1000 of cell culture containing about 10.sup.4
cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited
in each well of a 96-well, opaque-walled plate. 2. Control wells
were prepared containing medium and without cells. 3. ADC was added
to the experimental wells and incubated for 3-5 days. 4. The plates
were equilibrated to room temperature for approximately 30 minutes.
5. A volume of CellTiter-Glo Reagent equal to the volume of cell
culture medium present in each well was added. 6. The contents were
mixed for 2 minutes on an orbital shaker to induce cell lysis. 7.
The plate was incubated at room temperature for 10 minutes to
stabilize the luminescence signal. 8. Luminescence was recorded and
reported in graphs as RLU=relative luminescence units.
Example 19
Plasma Clearance in Rat
[1109] Plasma clearance pharmacokinetics of antibody drug
conjugates and total antibody was studied in Sprague-Dawley rats
(Charles River Laboratories, 250-275 gms each). Animals were dosed
by bolus tail vein injection (IV Push). Approximately 300 whole
blood was collected through jugular cannula, or by tail stick, into
lithium/heparin anticoagulant vessels at each timepoint: 0
(predose), 10, and 30 minutes; 1, 2, 4, 8, 24 and 36 hours; and 2,
3, 4, 7, 14, 21, 28 days post dose. Total antibody was measured by
ELISA--ECD/GxhuFc-HRP. Antibody drug conjugate was measured by
ELISA-MMAE/MMAF/ECD-Bio/SA-HRP.
Example 20
Plasma Clearance in Monkey
[1110] Plasma clearance pharmacokinetics of antibody drug
conjugates and total antibody can be studied in cynomolgus monkeys.
FIG. 12 shows a two-stage plasma concentration clearance study
after administration of H-MC-vc-MMAE to Cynomolgus monkeys at
different doses: 0.5, 1.5, 2.5, and 3.0 mg/kg, administered at day
1 and day 21. Concentrations of total antibody and ADC were
measured over time. (H=Trastuzumab).
Example 21
Tumor Volume In Vivo Efficacy in Transgenic Explant Mice
[1111] Animals suitable for transgenic experiments can be obtained
from standard commercial sources such as Taconic (Germantown,
N.Y.). Many strains are suitable, but FVB female mice are preferred
because of their higher susceptibility to tumor formation. FVB
males can be used for mating and vasectomized CD.1 studs can be
used to stimulate pseudopregnancy. Vasectomized mice can be
obtained from any commercial supplier. Founders can be bred with
either FVB mice or with 129/BL6.times.FVB p53 heterozygous mice.
The mice with heterozygosity at p53 allele can be used to
potentially increase tumor formation. Some F1 tumors are of mixed
strain. Founder tumors can be FVB only.
[1112] Animals having tumors (allograft propagated from Fo5 mmtv
transgenic mice) can be treated with a single or multiple dose by
IV injection of ADC. Tumor volume can be assessed at various time
points after injection.
Example 22
Synthesis of MC-MMAF via t-butyl ester
##STR00081##
[1114] MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 128.6 mg, 0.163
mmol) was suspended in CH.sub.2Cl.sub.2 (0.500 mL).
6-Maleimidocaproic acid (68.9 mg, 0.326 mmol) and
1,3-diisopropylcarbodiimide (0.0505 mL, 0.326 mmol) were added
followed by pyridine (0.500 mL). Reaction mixture was allowed to
stir for 1.0 hr. HPLC analysis indicated complete consumption of
starting compound 1. Volatile organics were evaporated under
reduced pressure. Product was isolated via flash column
chromatography, using a step gradient from 0 to 5% Methanol in
CH.sub.2Cl.sub.2. A total of 96 mg of pure
MC-MeVal-Val-Dil-Dap-Phe-OtBu (12) (60% yield) was recovered. ES-MS
m/z 981.26 [M+H].sup.+; 1003.47 [M+Na].sup.+; 979.65
[M-H].sup.-.
[1115] MC-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 12, 74 mg, 0.0754
mmol) was suspended in CH.sub.2Cl.sub.2 (2.0 mL) and TFA (1 mL) at
room temperature. After 2.5 hr, HPLC analysis indicated complete
consumption of starting material. Volatile organics were evaporated
under reduced pressure, and the product was isolated via
preparatory RP-HPLC, using a Phenomenex C.sub.12 Synergi Max-RP 80
.ANG. Column (250.times.21.20 mm). Eluent: linear gradient 10% to
90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90%
MeCN/0.05% TFA (aq) for an additional 20 minutes. ES-MS m/z 925.33
[M+H].sup.+; 947.30 [M+Na].sup.+; 923.45 [M-H].sup.-.
Example 23a
Synthesis of MC-MMAF (11) via dimethoxybenzyl ester
##STR00082## ##STR00083##
[1116] Preparation of Fmoc-L-Phenylalanine-2,4-dimethoxybenzyl
ester (Fmoc-Phe-ODMB)
[1117] A 3-neck, 5-L round-bottom flask was charged with
Fmoc-L-Phenylalanine (200 g, 516 mmol Bachem), 2,4-dimethoxybenzyl
alcohol (95.4 g, 567 mmol, Aldrich), and CH2Cl2 (2.0 L).
N,N-dimethylformamide t-butyl acetal (155 mL, 586 mmol, Fluka) was
added to the resulting suspension over 20 min under N2, which
resulted in a clear solution. The reaction was then stirred at room
temperature overnight, after which time TLC analysis (0.42,
Heptane/EtOAc=2:1) indicated that the reaction was complete. The
reaction mixture was concentrated under reduced pressure to give a
light yellow oil, which was redissolved in CH2Cl2 (200 mL) and
purified through a short plug of silica gel (25 cm.times.25 cm,
CH.sub.2Cl.sub.2) to give a colorless foam (250 g). MeCN (1 L) was
added into the resulting foam, which totally dissolved the batch.
It was then concentrated to dryness and redissolved in MeCN (1 L)
and the resulting suspension was stirred for 1 h, filtered and the
filter cake was rinsed with MeCN (2.times.200 mL) to give
Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester as a white solid
(113.58 g, 41%, 95.5% AUC by HPLC analysis). Data: HPLC.
Preparation L-Phenylalanine-2,4-dimethoxybenzyl ester
(Phe-ODMB)
[1118] A 500-mL round-bottom flask was charged with
Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester (26.00 g, 48.3
mmol), CH2Cl2 (150 mL) and diethylamine (75 mL, Acros). Mixture was
stirred at room temperature and the completion monitored by HPLC.
After 4 h, the mixture was concentrated (bath temp <30.degree.
C.). The residue was resuspended in CH2Cl2 (200 mL) and
concentrated. This was repeated once. To the residue was added MeOH
(20 mL), which caused the formation of a gel. This residue was
diluted with CH2Cl2 (200 mL), concentrated and the cloudy oil left
under vacuum overnight. The residue was suspended in CH2Cl2 (100
mL), then toluene (120 mL) was added. The mixture was concentrated
and the residue left under vacuum overnight.
[1119] Data: HPLC, .sup.1H NMR.
Preparation of Fmoc-Dolaproine (Fmoc-Dap)
[1120] Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HCl
in 1,4-dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5
hours, TLC analysis indicated the reaction was complete (10%
MeOH/CH2Cl2) and the mixture was concentrated to near-dryness.
Additional 1,4-dioxane was charged (50 mL) and the mixture was
concentrated to dryness and dried under vacuum overnight. The
resulting white solid was dissolved in H2O (400 mL) and transferred
to a 3-L, three-neck, round-bottom flask with a mechanical stirrer
and temperature probe. N,N-diisopropylethylamine (214.3 mL, 1.23
mol, Acros) was added over one minute, causing an exotherm from
20.5 to 28.2.degree. C. (internal). The mixture was cooled in an
ice bath and 1,4-dioxane was added (400 mL). A solution of Fmoc-OSu
(89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 mL) was
added from an addition funnel over 15 minutes, maintaining the
reaction temperature below 9.degree. C. The mixture was allowed to
warm to room temperature and stir for 19 hours, after which the
mixture was concentrated by rotary evaporation to an aqueous slurry
(390 g). The suspension was diluted with H2O (750 mL) and Et2O (750
mL), causing a copious white precipitate to form. The layers were
separated, keeping the solids with the organic layer. The aqueous
layer was acidified using conc. HCl (30 mL) and extracted with
EtOAc (3.times.500 mL). The combined extracts were dried over
MgSO4, filtered and concentrated to give 59.25 g of a yellow oil A.
The Et2O extract was extracted once with sat. NaHCO3 (200 mL),
keeping the solids with the aqueous layer. The aqueous suspension
was acidified using conc. HCl (50 mL) and extracted with Et2O (50
mL) keeping the solids with the organic layer. The organic layer
was filtered and concentrated to give 32.33 g of a yellow oil B.
The two oils (A and B) were combined and purified by flash
chromatography on silica gel eluting with CH2Cl2 (3.5 L), then 3%
MeOH/CH2Cl2 (9 L) to give 68.23 g of Fmoc-dolaproine as a white
foam (81%, 97.5% purity by HPLC (AUC)).
Preparation of Fmoc-Dap-Phe-ODMB
[1121] Crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF
(105 mL, Acros) for 5 minutes and Fmoc-Dap (19.80 g, 48.3 mmol) was
added. The mixture was cooled in an ice bath and TBTU (17.08 g,
53.20 mmol, Matrix Innovations) was added.
N,N-diisopropylethylamine (25.3 mL, 145.0 mmol, Acros) was added
via syringe over 3 min. After 1 h, the ice bath was removed and the
mixture was allowed to warm over 30 min. The mixture was poured
into water (1 L) and extracted with ethyl acetate (300 mL). After
separation, the aqueous layer was re-extracted with ethyl acetate
(2.times.150 mL). The combined organic layers were washed with
brine (150 mL), dried (MgSO4) and filtered (filter paper) to remove
the insolubles (inorganics and some dibenzofulvene). After
concentration, the residue (41 g) was adsorbed on silica (41 g) and
purified by chromatography (22 cm.times.8 cm column; 65%
Heptane/EtOAc (2.5 L); 33% Heptane/EtOAc (3.8 L), to give 29.4 g of
product as a white foam (86%, 92% purity by HPLC).
[1122] Data: HPLC, .sup.1H NMR, TLC (1:1 EtOAc/Heptane R.sub.f
0.33, red in vanillin stain).
Preparation of Dap-Phe-ODMB
[1123] A 1-L round bottom flask was charged with Fmoc-Dap-Phe-ODMB
(27.66 g), CH2Cl2 (122 mL) and diethylamine (61 mL, Acros). The
solution was stirred at room temperature and the completion
monitored by HPLC. After 7 h, the mixture was concentrated (bath
temp. <30.degree. C.). The residue was suspended in CH2Cl2 (300
mL) and concentrated. This was repeated twice. To the residue was
added MeOH (20 mL) and CH2Cl2 (300 mL), and the solution was
concentrated. The residue was suspended in CH2Cl2 (100 mL) and
toluene (400 mL), concentrated, and the residue left under vacuum
overnight to give a cream-like residue.
[1124] Data: HPLC, .sup.1H NMR, MS.
Preparation of Fmoc-Meval-Val-Dil-Dap-Phe-ODMB
[1125] Crude Dap-Phe-ODMB (39.1 mmol) was suspended in anhydrous
DMF (135 mL, Acros) for 5 minutes and Fmoc-MeVal-Val-Dil-OH (24.94
g, 39.1 mmol, see Example 2 for preparation) was added. The mixture
was cooled in an ice bath and TBTU (13.81 g, 43.0 mmol, Matrix
Innovations) was added. N,N-Diisopropylethylamine (20.5 mL, 117.3
mmol, Acros) was added via syringe over 2 minutes. After 1 hour,
the ice bath was removed and the mixture was allowed to warm over
30 min. The mixture was poured into water (1.5 L) and diluted with
ethyl acetate (480 mL). After standing for 15 minutes, the layers
were separated and the aqueous layer was extracted with ethyl
acetate (300 mL). The combined organic layers were washed with
brine (200 mL), dried (MgSO4) and filtered (filter paper) to remove
insolubles (inorganics and some dibenzofulvene). After
concentration, the residue (49 g) was scraped from the flask and
adsorbed on silica (49 g) and purified by chromatography (15
cm.times.10 cm dia column; 2:1 EtOAc/Heptane (3 L), EtOAc (5 L);
250 mL fractions) to give 31.84 g of
Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (73%, 93% purity by
HPLC (AUC)).
[1126] Data: HPLC, TLC (2:1 EtOAc/heptane, Rf=0.21, red in vanillin
stain).
Preparation of Meval-Val-Dil-Dap-Phe-ODMB
[1127] A 1-L, round-bottom flask was charged with
Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB (28.50 g), CH2Cl2 (80 mL) and
diethylamine (40 mL). Mixture was stirred at room temperature
overnight and then was concentrated under reduced pressure. The
residue was adsorbed on silica (30 g) and puffed by flash
chromatography (15 cm.times.8 cm dia column; 2% MeOH/DCM (2 L), 3%
MeOH/DCM (1 L), 6% MeOH/DCM (4 L); 250 mL fractions) to give 15.88
g of MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (69%, 96% purity by
HPLC (AUC)).
[1128] Data: HPLC, TLC (6% MeOH/DCM, Rf=0.24, red in vanillin
stain).
Preparation of Mc-Meval-Val-Dil-Dap-Phe-ODMB
[1129] A 50-mL, round-bottom flask was charged with
MeVal-Val-Dil-Dap-Phe-ODMB (750 mg, 0.85 mmol), anhydrous DMF (4
mL), maleimidocaproic acid (180 mg, 0.85 mmol), and TBTU (300 mg,
0.93 mmol, Matrix Innovations) at room temperature.
N,N-Diisopropylethylamine (450 .mu.L, 2.57 mmol) was added via
syringe. After 1.5 hours, the mixture was poured in water (50 mL)
and diluted with ethyl acetate (30 mL). NaCl was added to improve
the separation. After separation of the layers, the aqueous layer
was extracted with ethyl acetate (25 mL). The combined organic
layers were dried (MgSO4), filtered and concentrated. The resulting
oil (1 g) was purified by flash chromatography [100 mL silica; 25%
Heptane/EtOAc (100 mL), 10% Heptane/EtOAc (200 mL), EtOAc (1.5 L)]
to give MC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg,
57%, 94% purity by HPLC(AUC)).
[1130] Data: .sup.1H NMR, HPLC.
Preparation of MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF)(11)
[1131] A 50-mL, round-bottom flask was charged with
MC-MeVal-Val-Dil-Dap-Phe-ODMB (Compound 13, 428 mg, 0.39 mmol) and
dissolved in 2.5% TFA/CH.sub.2Cl.sub.2 (20 mL). The solution turned
pink-purple over 2 min. The completion was monitored by HPLC and
TLC (6% MeOH/DCM, KMnO4 stain). After 40 min, three drops of water
were added and the cloudy pink-purple mixture was concentrated to
give 521 mg of a pink residue. Purification by chromatography (15%
IPA/DCM) gave 270 mg of MC-MMAF (73%, 92% purity by HPLC) as a
white solid.
Example 23b
Synthesis of Analog of mc-MMAF
##STR00084##
[1133] MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 35 mg, 0.044 mmol)
was suspended in DMF (0.250 mL).
4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic acid (11 mg, 0.049
mmol) and HATU (17 mg, 0.044 mmol) were added followed by DIEA
(0.031 mL, 0.17 mmol). This reaction mixture was allowed to stir
for 2.0 hr. HPLC analysis indicated complete consumption of
starting compound 1.
[1134] Product was isolated via preparatory RP-HPLC, using a
Phenomenex C.sub.12 Synergi Max-RP 80 .ANG. Column (250.times.21.20
mm). Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8
minutes, then isocratic 80% MeCN/0.05% TFA (aq) for an additional
12 minutes. A total of 20 mg of pure product (14) was isolated
(0.02 mmol, 46% yield). ES-MS m/z 987.85 [M+H].sup.+; 1019.41
[M+Na].sup.+; 985.54 [M-H].sup.-.
[1135] MB-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 14, 38 mg, 0.0385
mmol) was suspended in CH.sub.2Cl.sub.2 (1 mL) and TFA (1 mL).
Mixture was stirred for 2.0 hr, and then volatile organics were
evaporated under reduced pressure. Product was purified by
preparatory RP-HPLC, using a Phenomenex C.sub.12 Synergi Max-RP 80
.ANG. Column (250.times.21.20 mm). Eluent: linear gradient 10% to
80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80%
MeCN/0.05% TFA (aq) for an additional 12 minutes. A total of 14.4
mg of MB-MMAF product was isolated (0.015 mmol, 40% yield). ES-MS
m/z 930.96 [M+H].sup.+952.98 [M+Na].sup.+; 929.37 [M-H].sup.-.
Example 23c
Preparation of MC-MeVal-Cit-PAB-MMAF (16)
##STR00085##
[1137] To a room temperature suspension of Fmoc-MeVal-OH (3.03 g,
8.57 mmol) and N,N'-disuccimidyl carbonate (3.29 g, 12.86 mmol) in
CH.sub.2Cl.sub.2 (80 mL) was added DIEA (4.48 mL, 25.71 mmol). This
reaction mixture was allowed to stir for 3.0 hr, and then poured
into a separation funnel where the organic mixture was extracted
with 0.1 M HCl (aq). The crude organic residue was concentrated
under reduced pressure, and the product was isolated by flash
column chromatography on silica gel using a 20-100% ethyl
acetate/hexanes linear gradient. A total of 2.18 g of pure
Fmoc-MeVal-OSu (4.80 mmoles, 56% yield) was recovered.
[1138] To a room temperature suspension of Fmoc-MeVal-OSu (2.18 g,
4.84 mmol) in DME (13 mL) and THF (6.5 mL) was added a solution of
L-citrulline (0.85 g, 4.84 mmol) and NaHCO.sub.3 (0.41 g, 4.84
mmol) in H.sub.2O (13 mL). The suspension was allowed to stir at
room temperature for 16 hr, then it was extracted into
tert-BuOH/CHCl.sub.3/H.sub.2O, acidified to pH=2-3 with 1 M HCl.
The organic phase was separated, dried and concentrated under
reduced pressure. The residue was triturated with diethyl ether
resulting in 2.01 g of Fmoc-MeVal-Cit-COOH which was used without
further purification.
[1139] The crude Fmoc-MeVal-Cit-COOH was suspended in 2:1
CH.sub.2Cl.sub.2/MeOH (100 mL), and to it was added p-aminobenzyl
alcohol (0.97 g, 7.9 mmol) and EEDQ (1.95 g, 7.9 mmol). This
suspension was allowed to stir for 125 hr, then the volatile
organics were removed under reduced pressure, and the residue was
purified by flash column chromatography on silica gel using a 10%
MeOH/CH.sub.2Cl.sub.2. Pure Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896
mmol, 18.5% yield) was recovered. ES-MS m/z 616.48 [M+H].sup.+.
[1140] To a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896
mmol) in CH.sub.2Cl.sub.2 (40 mL) was added STRATOSPHERES.TM.
(piperizine-resin-bound) (>5 mmol/g, 150 mg). After being
stirred at room temperature for 16 hr the mixture was filtered
through celite (pre-washed with MeOH), and concentrated under
reduced pressure. Residue was triturated with diethyl ether and
hexanes. Resulting solid material, MeVal-Cit-PAB-OH, was suspended
in CH.sub.2Cl.sub.2 (20 mL), and to it was added MC-OSu (0.28 g,
0.896 mmol), DIEA (0.17 mL, 0.99 mmol), and DMF (15 mL). This
suspension was stirred for 16 hr, but HPLC analysis of the reaction
mixture indicated incomplete reaction, so the suspension was
concentrated under reduced pressure to a volume of 6 mL, then a 10%
NaHCO.sub.3 (aq) solution was added and the suspension stirred for
an additional 16 hr. Solvent was removed under reduced pressure,
and the residue was purified by flash column chromatography on
silica gel using a 0-10% MeOH/CH.sub.2Cl.sub.2 gradient, resulting
in 42 mg (0.072 mmol, 8% yield) of MC-MeVal-Cit-PAB-OH.
[1141] To a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol)
and bis(nitrophenyl)carbonate (2.59 g, 8.52 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added DIEA (1.06 mL, 6.06 mmol). This
suspension was stirred for 5.5 hr, concentrated under reduced
pressure and purified by trituration with diethyl ether.
MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) was suspended in a
1:5 pyridine/DMF solution (3 mL), and to it was added HOBt (5 mg,
0.039 mmol), DIEA (0.17 mL, 0.978 mmol) and MMAF (compound 2, 150
mg, 0.205 mmol). This reaction mixture was stirred for 16 hr at
room temperature, and then purified by preparatory RP-HPLC
(.times.3), using a Phenomenex C.sub.12 Synergi Max-RP 80 .ANG.
Column (250.times.21.20 mm). Eluent: linear gradient 10% to 90%
MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90% MeCN/0.05%
TFA (aq) for an additional 20 minutes. MC-MeVal-Cit-PAB-MMAF (16)
was obtained as a yellowish solid (24.5 mg, 0.0182, 0.45% yield).
ES-MS m/z 1344.95 [M+H].sup.+; 1366.94 [M+Na].sup.+.
Example 23c
Preparation of succinimide ester of suberyl-Val-Cit-PAB-MMAF
(17)
##STR00086##
[1143] Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436
mg, 0.57 mmol, 1.5 eq.) were suspended in anhydrous pyridine, 5 mL.
HOBt (10 mg, 0.076 mmol, 0.2 eq.) was added followed by DIEA (199
p. 1, 1.14 mmol, 3 eq.). Reaction mixture was sonicated for 10 min,
and then stirred overnight at room temperature. Pyridine was
removed under reduced pressure, residue was re-suspended in
CH.sub.2Cl.sub.2. Mixture was separated by silica gel flash
chromatography in a step gradient of MeOH, from 0 to 10%, in
CH.sub.2Cl.sub.2. Product containing fractions were pulled,
concentrated, dried in vacuum overnight to give 317 mg (59% yield)
of Fmoc-Val-Cit-PAB-MMAF-OtBu. ES-MS m/z 1415.8 [M+H].sup.+.
[1144] Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20%
TFA/CH.sub.2Cl.sub.2 (10 mL), for 2 hrs. Mixture was diluted with
CH.sub.2Cl.sub.2 (50 mL). Organic layer was washed successively
with water (2.times.30 mL) and brine (1.times.30 mL). Organic phase
was concentrated, loaded onto pad of silica gel in 10%
MeOH/CH.sub.2Cl.sub.2. Product was eluted with 30%
MeOH/CH.sub.2Cl.sub.2. After drying in vacuum overnight,
Fmoc-Val-Cit-PAB-MMAF was obtained as a white solid, 38 mg, 40%
yield. ES-MS m/z 1357.7 [M-H].sup.-.
[1145] Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in
CH.sub.2Cl.sub.2 (2 mL) diethylamine (2 mL) and DMF (2 mL). Mixture
was stirred for 2 hrs at room temperature. Solvent was removed
under reduced pressure. Residue was co-evaporated with pyridine (2
mL), then with toluene (2.times.5 mL), dried in vacuum.
Val-Cit-PAB-MMAF was obtained as brownish oil, and used without
further purification.
[1146] All Val-Cit-PAB-MMAF prepared from 67 mg of
Fmoc-Val-Cit-PAB-MMAF, was suspended in pyridine (2 mL), and added
to a solution of disuccinimidyl suberate (74 mg, 0.2 mmol, 4 eq.),
in pyridine (1 mL). Reaction mixture was stirred at room
temperature. After 3 hrs ether (20 mL) was added. Precipitate was
collected, washed with additional amount of ether. Reddish solid
was suspended in 30% MeOH/CH.sub.2Cl.sub.2, filtered through a pad
of silica gel with 30% MeOH/CH.sub.2Cl.sub.2 as an eluent. Compound
17 was obtained as white solid, 20 mg (29% yield). ES-MS m/z 1388.5
[M-H].sup.-
Example 24
In Vivo Efficacy of mcMMAF Antibody-Drug Conjugates
[1147] Efficacy of cAC10-mcMMAF in Karpas-299 ALCL xenografts: To
evaluate the in vivo efficacy of cAC10-mcMMAF with an average of 4
drug moieties per antibody (cAC10-mcF4), Karpas-299 human ALCL
cells were implanted subcutaneously into immunodeficient C.B-17
SCID mice (5.times.10.sup.6 cells per mouse). Tumor volumes were
calculated using the formula (0.5.times.L.times.W.sup.2) where L
and W are the longer and shorter of two bidirectional measurements.
When the average tumor volume in the study animals reached
approximately 100 mm.sup.3 (range 48-162) the mice were divided
into 3 groups (5 mice per group) and were either left untreated or
were given a single intravenous injection through the tail vein of
either 1 or 2 mg/kg cAC10-mcF4 (FIG. 1). The tumors in the
untreated mice grew rapidly to an average volume of >1,000
mm.sup.3 within 7 days of the start of therapy. In contrast, all of
the cAC10-mcF4 treated tumor showed rapid regression with 3/5 in
the 1 mg/kg group and 5/5 in the 2 mg/kg group obtaining complete
tumor response. While the tumor in one of the complete responders
in the 2 mg/kg group did recur approximately 4 weeks later, there
were no detectable tumors in the remaining 4/5 responders in this
group and in the 3 complete responders in the 1 mg/kg group at 10
weeks post therapy.
[1148] Efficacy of cBR96-mcMMAF in L2987 NSCLC xenografts: cBR96 is
a chimeric antibody that recognizes the Le.sup.Y antigen. To
evaluate the in vivo efficacy of cBR96-mcMMAF with 4 drugs per
antibody (cBR96-mcF4) L2987 non-small cell lung cancer (NSCLC)
tumor fragments were implanted into athymic nude mice. When the
tumors averaged approximately 100 mm.sup.3 the mice were divided
into 3 groups: untreated and 2 therapy groups. For therapy, as
shown in FIG. 3a, mice were administered cBR96-mcF4 at either 3 or
10 mg/kg/injection every 4 days for a total of 4 injections
(q4d.times.4). As shown in FIG. 3b, mice were administered
cBR96-mcF4 or a non-binding control conjugate, cAC 10-mcF4, at 10
mg/kg/injection every 4 days for a total of 4 injections
(q4d.times.4). As shown in FIGS. 3a and 3b, BR96-mcF4 produced
pronounced tumor growth delay compared to the controls.
[1149] FIG. 2 shows an in vivo, single dose, efficacy assay of
cAC10-mcMMAF in subcutaneous L540CY. For this study there were 4
mice in the untreated group and 10 in each of the treatment
groups.
Example 25
In Vitro Efficacy of MC-MMAF Antibody-Drug Conjugates
[1150] Activity of cAC10-antibody-drug conjugates against
CD30.sup.+ cell lines. FIGS. 4a and 16b show dose-response curves
from a representative experiment where cultures of Karpas 299
(anaplastic large cell lymphoma) and L428 (Hodgkin's Lymphoma) were
incubated with serially diluted cAC10-mcMMAF (FIG. 4a) or
cAC10-vcMMAF (FIG. 4b) for 96 hours. The cultures were labeled for
4 hours with 50 .mu.M resazurin [7-hydroxy-3H-phenoxazin-3-one
10-oxide] and the fluorescence measured. The data were reduced in
GraphPad Prism version 4.00 using the 4-parameter dose-response
curve fit procedure. IC.sub.50 values are defined as the
concentration where growth is reduced 50% compared with untreated
control cultures. Each concentration was tested in
quadruplicate.
[1151] Activity of cBR96-antibody-drug conjugates against Le.sup.y+
cell lines. FIGS. 5a and 5b show dose-response curves from a
representative experiment where cultures of H3396 (breast
carcinoma) and L2987 (non small cell lung carcinoma) were incubated
with serially diluted cBR96-mcMMAF (FIG. 5a) or -veMMAF (FIG. 5b)
for 96 hours. The cultures were labeled for 4 hours with 50 .mu.M
resazurin and the fluorescence measured. The data were reduced in
GraphPad Prism version 4.00 using the 4-parameter dose-response
curve fit procedure. IC.sub.50 values are defined as the
concentration where growth is reduced 50% compared with untreated
control cultures. Each concentration is tested in
quadruplicate.
[1152] Activity of c1F6-antibody-drug conjugates against CD70.sup.+
renal cell carcinoma cell lines. FIGS. 6a and 6b show dose-response
curves from a representative experiment where cultures of Caki-1
and 786-0 cells were incubated with serially diluted c1F6-mcMMAF
(FIG. 6a) or -vcMMAF (FIG. 6b) for 96 hours. The cultures were
labeled for 4 hours with 50 .mu.M resazurin and the fluorescence
measured. The data were reduced in GraphPad Prism version 4.00
using the 4-parameter dose-response curve fit procedure. IC.sub.50
values are defined as the concentration where growth is reduced 50%
compared with untreated control cultures. Each concentration is
tested in quadruplicate.
Example 26
Purification of Trastuzumab
[1153] One vial containing 440 mg HERCEPTIN.RTM. (huMAb4D5-8,
rhuMAb HER2, U.S. Pat. No. 5,821,337) antibody) was dissolved in 50
mL MES buffer (25 mM MES, 50 mM NaCl, pH 5.6) and loaded on a
cation exchange column (Sepharose S, 15 cm.times.1.7 cm) that had
been equilibrated in the same buffer. The column was then washed
with the same buffer (5 column volumes). Trastuzumab was eluted by
raising the NaCl concentration of the buffer to 200 mM. Fractions
containing the antibody were pooled, diluted to 10 mg/mL, and
dialyzed into a buffer containing 50 mm potassium phosphate, 50 mM
NaCl, 2 mM EDTA, pH 6.5.
Example 27
Preparation of Trastuzumab-MC-MMAE by Conjugation of Trastuzumab
and MC-MMAE
[1154] Trastuzumab, dissolved in 500 mM sodium borate and 500 mM
sodium chloride at pH 8.0 is treated with an excess of 100 mM
dithiothreitol (DTT). After incubation at 37.degree. C. for about
30 minutes, the buffer is exchanged by elution over Sephadex G25
resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is
checked by determining the reduced antibody concentration from the
absorbance at 280 nm of the solution and the thiol concentration by
reaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of
the absorbance at 412 nm. The reduced antibody dissolved in PBS is
chilled on ice.
[1155] The drug linker reagent, maleimidocaproyl-monomethyl
auristatin E (MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in
acetonitrile and water at known concentration, and added to the
chilled reduced antibody trastuzumab in PBS. After about one hour,
an excess of maleimide is added to quench the reaction and cap any
unreacted antibody thiol groups. The reaction mixture is
concentrated by centrifugal ultrafiltration and trastuzumab-MC-MMAE
is purified and desalted by elution through G25 resin in PBS,
filtered through 0.2 .mu.m filters under sterile conditions, and
frozen for storage.
Example 28
Preparation of Trastuzumab-MC-MMAF by Conjugation of Trastuzumab
and MC-MMAF
[1156] Trastuzumab-MC-MMAF was prepared by conjugation of
trastuzumab and MC-MMAF following the procedure of Example 27.
Example 29
Preparation of Trastuzumab-MC-val-cit-PAB-MMAE by Conjugation of
Trastuzumab and MC-val-cit-PAB-MMAE
[1157] Trastuzumab-MC-val-cit-PAB-MMAE was prepared by conjugation
of trastuzumab and MC-val-cit-PAB-MMAE following the procedure of
Example 27.
Example 30
Preparation of trastuzumab-MC-val-cit-PAB-MMAF by Conjugation of
trastuzumab and MC-val-cit-PAB-MMAF 9
[1158] Trastuzumab-MC-val-cit-PAB-MMAF was prepared by conjugation
of trastuzumab and MC-val-cit-PAB-MMAF 9 following the procedure of
Example 27.
Example 31
Rat Toxicity
[1159] The acute toxicity profile of free drugs and ADC was
evaluated in adolescent Sprague-Dawley rats (75-125 gms each,
Charles River Laboratories (Hollister, Calif.). Animals were
injected on day 1, complete chemistry and hematology profiles were
obtained at baseline, day 3 and day 5 and a complete necropsy was
performed on day 5. Liver enzyme measurements was done on all
animals and routine histology as performed on three random animals
for each group for the following tissues: sternum, liver, kidney,
thymus, spleen, large and small intestine. The experimental groups
were as follows:
TABLE-US-00052 .mu.g MMAF/ MMAF/ Group Administered mg/kg m.sup.2
MAb N/Sex 1 Vehicle 0 0 0 2/F 2 trastuzumab-MC-val-cit- 9.94 840
4.2 6/F MMAF 3 trastuzumab-MC-val-cit- 24.90 2105 4.2 6/F MMAF 4
trastuzumab-MC(Me)- 10.69 840 3.9 6/F val-cit-PAB-MMAF 5
trastuzumab-MC(Me)- 26.78 2105 3.9 6/F val-cit-PAB-MMAF 6
trastuzumab-MC-MMAF 10.17 840 4.1 6/F 7 trastuzumab-MC-MMAF 25.50
2105 4.1 6/F 8 trastuzumab-MC-val-cit- 21.85 2105 4.8 6/F
PAB-MMAF
[1160] For trastuzumab-MC-val-cit-MMAF,
trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and
trastuzumab-MC-val-cit-PAB-MMAF, the .mu.g MMAF/m.sup.2 was
calculated using 731.5 as the MW of MMAF and 145167 as the MW of
Herceptin.
[1161] The body surface area was calculated as follows: [{(body
weight in grams to 0.667 power).times.11.8}/10000]. (Guidance for
Industry and Reviewers, 2002).
[1162] The dose solutions were administered by a single intravenous
bolus tail-vein injection on Study Day 1 at a dose volume of 10
mL/kg. Body weights of the animals were measured pre-dose on Study
Day 1 and daily thereafter. Whole blood was collected into EDTA
containing tubes for hematology analysis. Whole blood was collected
into serum separator tubes for clinical chemistry analysis. Blood
samples were collected pre-dose on Study Day--4, Study Day 3 and
Study Day 5. Whole blood was also collected into sodium heparin
containing tubes at necropsy and the plasma was frozen at
-70.degree. C. for possible later analysis. The following tissues
were collected and placed in neutral buffered formalin at necropsy:
liver, kidneys, heart, thymus, spleen, brain, sternum and sections
of the GI tract, including stomach, large and small intestine.
Sternum, small intestine, large intestine, liver, thymus, spleen
and kidney were examined.
[1163] Liver associated serum enzyme levels at each timepoint were
compared to a range (5th and 95th percentile) from normal female
Sprague-Dawley rats. White blood cell and platelet counts at each
timepoint were compared to a range (5th and 95th percentile) from
normal female Sprague-Dawley rats.
[1164] High dose study in normal female Sprague-Dawley rats:
TABLE-US-00053 Group 1: Vehicle Group 2: trastuzumab-MC-MMAF, 52.24
mg/kg, 4210 .mu.g/m.sup.2 Group 3: trastuzumab-MC-MMAF, 68.25
mg/kg, 5500 .mu.g/m.sup.2 Group 4: trastuzumab-MC-MMAF, 86.00
mg/kg, 6930 .mu.g/m.sup.2
[1165] Tissues from 11 animals were submitted for routine
histology. These animals had been part of an acute dose-ranging
toxicity study using a trastuzumab-MC-MMAF immunoconjugate. Animals
were followed for 12 days following dosing.
Example 32
Cynomolgus Monkey Toxicity/Safety
[1166] Three groups of four (2 male, 2 female) naive Macaca
fascicularis (cynomolgus monkey) were studied for
trastuzumab-MC-vc-PAB-MMAE and trastuzumab-MC-vc-PAB-MMAF.
Intravenous administration was conducted at days 1 and 22 of the
studies.
TABLE-US-00054 Sample Group Dose Vehicle 1 day 1 1M/1F day 22
H-MC-vc-PAB-MMAE 2 180 .mu.g/m.sup.2 (0.5 mg/kg) at day 1 2M/2F
1100 .mu.g/m.sup.2 (3.0 mg/kg) at day 22 H-MC-vc-PAB-MMAE 3 550
.mu.g/m.sup.2 (1.5 mg/kg) at day 8 2M/2F 550 .mu.g/m.sup.2 (1.5
mg/kg) at day 29 H-MC-vc-PAB-MMAE 4 880 .mu.g/m.sup.2 (2.5 mg/kg)
at day 15 2M/2F 880 .mu.g/m.sup.2 (2.5 mg/kg) at day 36 Vehicle 1
day 1 1M/1F day 22 H-MC-vc-PAB-MMAF 2 180 .mu.g/m.sup.2 (0.5 mg/kg)
at day 1 2M/2F 1100 .mu.g/m.sup.2 (3.0 mg/kg) at day 22
H-MC-vc-PAB-MMAF 3 550 .mu.g/m.sup.2 (1.5 mg/kg) at day 1 2M/2F 550
.mu.g/m.sup.2 (1.5 mg/kg) at day 22 H-MC-vc-PAB-MMAF 4 880
.mu.g/m.sup.2 (2.5 mg/kg) at day 1 2M/2F 880 .mu.g/m.sup.2 (2.5
mg/kg) at day 22 H = trastuzumab
[1167] Dosing is expressed in surface area of an animal so as to be
relevant to other species, i.e. dosage at .mu.g/m.sup.2 is
independent of species and thus comparable between species.
Formulations of ADC contained PBS, 5.4 mM sodium phosphate, 4.2 mM
potassium phosphate, 140 mM sodium chloride, pH 6.5.
[1168] Blood was collected for hematology analysis predose, and at
5 min., 6 hr, 10 hr, and 1, 3, 5, 7, 14, 21 days after each dose.
Erythrocyte (RBC) and platelet (PLT) counts were measured by the
light scattering method. Leukocyte (WBC) count was measured by the
peroxidase/basophil method. Reticulocyte count was measured by the
light scattering method with cationic dye. Cell counts were
measured on an Advia 120 apparatus. ALT (alanine aminotransferase)
and AST (aspartate aminotransferase) were measured in U/L by
UV/NADH; IFCC methodology on an Olympus AU400 apparatus, and using
Total Ab ELISA-ECD/GxhuFc-HRP. Conj. Ab
ELISA-MMAE/MMAF//ECD-Bio/SA-HRP tests.
Example 33
Production, Characterization and Humanization of Anti-ErbB2
Monoclonal Antibody 4D5
[1169] The murine monoclonal antibody 4D5 which specifically binds
the extracellular domain of ErbB2 was produced as described in
Fendly et al. (1990) Cancer Research 50:1550-1558. Briefly, NIH
3T3/HER2-3.sub.400 cells (expressing approximately 1.times.10.sup.5
ErbB2 molecules/cell) produced as described in Hudziak et al. Proc.
Natl. Acad. Sci. (USA) 84:7158-7163 (1987) were harvested with
phosphate buffered saline (PBS) containing 25 mM EDTA and used to
immunize BALB/c mice. The mice were given injections i.p. of
10.sup.7 cellsin 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice with
antisera that immunoprecipitated .sup.32P-labeled ErbB2 were given
i.p. injections of a wheat germ agglutinin-Sepharose (WGA) purified
ErbB2 membrane extract on weeks 9 and 13. This was followed by an
i.v. injection of 0.1 ml of the ErbB2 preparation and the
splenocytes were fused with mouse myeloma line X63-Ag8.653.
Hybridoma supernatants were screened for ErbB2-binding by ELISA and
radioimmunoprecipitation.
Epitope Mapping and Characterization
[1170] The ErbB2 epitope bound by monoclonal antibody 4D5 was
determined by competitive binding analysis (Fendly et al. Cancer
Research 50:1550-1558 (1990)). Cross-blocking studies were done by
direct fluorescence on intact cells using the PANDEXTM Screen
Machine to quantitate fluorescence. The monoclonal antibody was
conjugated with fluorescein isothiocyanate (FITC), using
established procedures (Wofsy et al. Selected Methods in Cellular
Immunology, p. 287, Mishel and Schiigi (eds.) San Francisco: W.J.
Freeman Co. (1980)). Confluent monolayers of NIH 3T3/HER2-3.sub.400
cells were trypsinized, washed once, and resuspended at
1.75.times.10.sup.6 cell/ml in cold PBS containing 0.5% bovine
serum albumin (BSA) and 0.1% NaN.sub.3. A final concentration of 1%
latex particles (IDC, Portland, Oreg.) was added to reduce clogging
of the PANDEX.TM. plate membranes. Cells in suspension, 20 and 20
.mu.l of purified monoclonal antibodies (100 .mu.g/ml to 0.1
.mu.g/ml) were added to the PANDEX.TM. plate wells and incubated on
ice for 30 minutes. A predetermined dilution of the FITC-labeled
monoclonal antibody in 20 .mu.l was added to each well, incubated
for 30 minutes, washed, and the fluorescence was quantitated by the
PANDEX.TM. Monoclonal antibodies were considered to share an
epitope if each blocked binding of the other by 50% or greater in
comparison to an irrelevant monoclonal antibody control. In this
experiment, monoclonal antibody 4D5 was assigned epitope I (amino
acid residues from about 529 to about 625, inclusive within the
ErbB2 extracellular domain.
[1171] The growth inhibitory characteristics of monoclonal antibody
4D5 were evaluated using the breast tumor cell line, SK-BR-3 (see
Hudziak et al. (1989) Molec. Cell. Biol. 9(3):1165-1172). Briefly,
SK-BR-3 cells were detached by using 0.25% (vol/vol) trypsin and
suspended in complete medium at a density of 4.times.10.sup.5 cells
per ml. Aliquots of 100 .mu.l (4.times.10.sup.4 cells) were plated
into 96-well microdilution plates, the cells were allowed to
adhere, and 100 .mu.l of media alone or media containing monoclonal
antibody (final concentration 5 .mu.g/ml) was then added. After 72
hours, plates were washed twice with PBS (pH 7.5), stained with
crystal violet (0.5% in methanol), and analyzed for relative cell
proliferation as described in Sugarman et al. (1985) Science
230:943-945. Monoclonal antibody 4D5 inhibited SK-BR-3 relative
cell proliferation by about 56%.
[1172] Monoclonal antibody 4D5 was also evaluated for its ability
to inhibit HRG-stimulated tyrosine phosphorylation of proteins in
the M.sub.r 180,000 range from whole-cell lysates of MCF7 cells
(Lewis et al. (1996) Cancer Research 56:1457-1465). MCF7 cells are
reported to express all known ErbB receptors, but at relatively low
levels. Since ErbB2, ErbB3, and ErbB4 have nearly identical
molecular sizes, it is not possible to discern which protein is
becoming tyrosine phosphorylated when whole-cell lysates are
evaluated by Western blot analysis. However, these cells are ideal
for HRG tyrosine phosphorylation assays because under the assay
conditions used, in the absence of exogenously added HRG, they
exhibit low to undetectable levels of tyrosine phosphorylation
proteins in the M.sub.r 180,000 range.
[1173] MCF7 cells were plated in 24-well plates and monoclonal
antibodies to ErbB2 were added to each well and incubated for 30
minutes at room temperature; then rHRG.beta.1.sub.177-244 was added
to each well to a final concentration of 0.2 nM, and the incubation
was continued for 8 minutes. Media was carefully aspirated from
each well, and reactions were stopped by the addition of 100 .mu.l
of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH
6.8). Each sample (25 .mu.l) was electrophoresed on a 4-12%
gradient gel (Novex) and then electrophoretically transferred to
polyvinylidene difluoride membrane. Antiphosphotyrosine (4G10, from
UBI, used at 1 .mu.g/ml) immunoblots were developed, and the
intensity of the predominant reactive band at {tilde over
(M)}.sub.r180,000 was quantified by reflectance densitometry, as
described previously (Holmes et al. (1992) Science 256:1205-1210;
Sliwkowski et al. J. Biol. Chem. 269:14661-14665 (1994)).
[1174] Monoclonal antibody 4D5 significantly inhibited the
generation of a HRG-induced tyrosine phosphorylation signal at
M.sub.r 180,000. In the absence of HRG, but was unable to stimulate
tyrosine phosphorylation of proteins in the M.sub.r 180,000 range.
Also, this antibody does not cross-react with EGFR (Fendly et al.
Cancer Research 50:1550-1558 (1990)), ErbB3, or ErbB4. Monoclonal
antibody 4D5 was able to block HRG stimulation of tyrosine
phosphorylation by 50%.
[1175] The growth inhibitory effect of monoclonal antibody 4D5 on
MDA-MB-175 and SK-BR-3 cells in the presence or absence of
exogenous rHRG.beta.1 was assessed (Schaefer et al. Oncogene
15:1385-1394 (1997)). ErbB2 levels in MDA-MB-175 cells are 4-6
times higher than the level found in normal breast epithelial cells
and the ErbB2-ErbB4 receptor is constitutively tyrosine
phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 was
able to inhibit cell proliferation of MDA-MB-175 cells, both in the
presence and absence of exogenous HRG. Inhibition of cell
proliferation by 4D5 is dependent on the ErbB2 expression level
(Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993)). A
maximum inhibition of 66% in SK-BR-3 cells could be detected.
However this effect could be overcome by exogenous HRG.
[1176] The murine monoclonal antibody 4D5 was humanized, using a
"gene conversion mutagenesis" strategy, as described in U.S. Pat.
No. 5,821,337, the entire disclosure of which is hereby expressly
incorporated by reference. The humanized monoclonal antibody 4D5
used in the following experiments is designated huMAb4D5-8. This
antibody is of IgG1 isotype.
REFERENCES CITED
[1177] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples which are intended
as illustrations of a few aspects of the invention and any
embodiments that are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the art and are intended to fall
within the scope of the appended claims.
[1178] All references cited herein are incorporated by reference in
their entirety and for all purposes to the same extent as if each
individual publication or patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 35 <210> SEQ ID NO 1 <211> LENGTH: 502 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: BMPR1B <400> SEQUENCE: 1 Met
Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys Lys Glu 1 5 10
15 Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys Val Leu Arg Cys
20 25 30 Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn Ile
Cys Ser 35 40 45 Thr Asp Gly Tyr Cys Phe Thr Met Ile Glu Glu Asp
Asp Ser Gly Leu 50 55 60 Pro Val Val Thr Ser Gly Cys Leu Gly Leu
Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Arg Asp Thr Pro Ile Pro His
Gln Arg Arg Ser Ile Glu Cys Cys 85 90 95 Thr Glu Arg Asn Glu Cys
Asn Lys Asp Leu His Pro Thr Leu Pro Pro 100 105 110 Leu Lys Asn Arg
Asp Phe Val Asp Gly Pro Ile His His Arg Ala Leu 115 120 125 Leu Ile
Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile Ile Leu 130 135 140
Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser 145
150 155 160 Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile Pro Pro Gly Glu
Ser Leu 165 170 175 Arg Asp Leu Ile Glu Gln Ser Gln Ser Ser Gly Ser
Gly Ser Gly Leu 180 185 190 Pro Leu Leu Val Gln Arg Thr Ile Ala Lys
Gln Ile Gln Met Val Lys 195 200 205 Gln Ile Gly Lys Gly Arg Tyr Gly
Glu Val Trp Met Gly Lys Trp Arg 210 215 220 Gly Glu Lys Val Ala Val
Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 225 230 235 240 Trp Phe Arg
Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu 245 250 255 Asn
Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp 260 265
270 Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr
275 280 285 Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala Lys Ser Met Leu
Lys Leu 290 295 300 Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu His
Thr Glu Ile Phe 305 310 315 320 Ser Thr Gln Gly Lys Pro Ala Ile Ala
His Arg Asp Leu Lys Ser Lys 325 330 335 Asn Ile Leu Val Lys Lys Asn
Gly Thr Cys Cys Ile Ala Asp Leu Gly 340 345 350 Leu Ala Val Lys Phe
Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro 355 360 365 Asn Thr Arg
Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val Leu Asp 370 375 380 Glu
Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile Met Ala Asp Met 385 390
395 400 Tyr Ser Phe Gly Leu Ile Leu Trp Glu Val Ala Arg Arg Cys Val
Ser 405 410 415 Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr His Asp
Leu Val Pro 420 425 430 Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu Ile
Val Cys Ile Lys Lys 435 440 445 Leu Arg Pro Ser Phe Pro Asn Arg Trp
Ser Ser Asp Glu Cys Leu Arg 450 455 460 Gln Met Gly Lys Leu Met Thr
Glu Cys Trp Ala His Asn Pro Ala Ser 465 470 475 480 Arg Leu Thr Ala
Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu 485 490 495 Ser Gln
Asp Ile Lys Leu 500 <210> SEQ ID NO 2 <211> LENGTH: 507
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: SLC7A5
<400> SEQUENCE: 2 Met Ala Gly Ala Gly Pro Lys Arg Arg Ala Leu
Ala Ala Pro Ala Ala 1 5 10 15 Glu Glu Lys Glu Glu Ala Arg Glu Lys
Met Leu Ala Ala Lys Ser Ala 20 25 30 Asp Gly Ser Ala Pro Ala Gly
Glu Gly Glu Gly Val Thr Leu Gln Arg 35 40 45 Asn Ile Thr Leu Leu
Asn Gly Val Ala Ile Ile Val Gly Thr Ile Ile 50 55 60 Gly Ser Gly
Ile Phe Val Thr Pro Thr Gly Val Leu Lys Glu Ala Gly 65 70 75 80 Ser
Pro Gly Leu Ala Leu Val Val Trp Ala Ala Cys Gly Val Phe Ser 85 90
95 Ile Val Gly Ala Leu Cys Tyr Ala Glu Leu Gly Thr Thr Ile Ser Lys
100 105 110 Ser Gly Gly Asp Tyr Ala Tyr Met Leu Glu Val Tyr Gly Ser
Leu Pro 115 120 125 Ala Phe Leu Lys Leu Trp Ile Glu Leu Leu Ile Ile
Arg Pro Ser Ser 130 135 140 Gln Tyr Ile Val Ala Leu Val Phe Ala Thr
Tyr Leu Leu Lys Pro Leu 145 150 155 160 Phe Pro Thr Cys Pro Val Pro
Glu Glu Ala Ala Lys Leu Val Ala Cys 165 170 175 Leu Cys Val Leu Leu
Leu Thr Ala Val Asn Cys Tyr Ser Val Lys Ala 180 185 190 Ala Thr Arg
Val Gln Asp Ala Phe Ala Ala Ala Lys Leu Leu Ala Leu 195 200 205 Ala
Leu Ile Ile Leu Leu Gly Phe Val Gln Ile Gly Lys Gly Val Val 210 215
220 Ser Asn Leu Asp Pro Asn Phe Ser Phe Glu Gly Thr Lys Leu Asp Val
225 230 235 240 Gly Asn Ile Val Leu Ala Leu Tyr Ser Gly Leu Phe Ala
Tyr Gly Gly 245 250 255 Trp Asn Tyr Leu Asn Phe Val Thr Glu Glu Met
Ile Asn Pro Tyr Arg 260 265 270 Asn Leu Pro Leu Ala Ile Ile Ile Ser
Leu Pro Ile Val Thr Leu Val 275 280 285 Tyr Val Leu Thr Asn Leu Ala
Tyr Phe Thr Thr Leu Ser Thr Glu Gln 290 295 300 Met Leu Ser Ser Glu
Ala Val Ala Val Asp Phe Gly Asn Tyr His Leu 305 310 315 320 Gly Val
Met Ser Trp Ile Ile Pro Val Phe Val Gly Leu Ser Cys Phe 325 330 335
Gly Ser Val Asn Gly Ser Leu Phe Thr Ser Ser Arg Leu Phe Phe Val 340
345 350 Gly Ser Arg Glu Gly His Leu Pro Ser Ile Leu Ser Met Ile His
Pro 355 360 365 Gln Leu Leu Thr Pro Val Pro Ser Leu Val Phe Thr Cys
Val Met Thr 370 375 380 Leu Leu Tyr Ala Phe Ser Lys Asp Ile Phe Ser
Val Ile Asn Phe Phe 385 390 395 400 Ser Phe Phe Asn Trp Leu Cys Val
Ala Leu Ala Ile Ile Gly Met Ile 405 410 415 Trp Leu Arg His Arg Lys
Pro Glu Leu Glu Arg Pro Ile Lys Val Asn 420 425 430 Leu Ala Leu Pro
Val Phe Phe Ile Leu Ala Cys Leu Phe Leu Ile Ala 435 440 445 Val Ser
Phe Trp Lys Thr Pro Val Glu Cys Gly Ile Gly Phe Thr Ile 450 455 460
Ile Leu Ser Gly Leu Pro Val Tyr Phe Phe Gly Val Trp Trp Lys Asn 465
470 475 480 Lys Pro Lys Trp Leu Leu Gln Gly Ile Phe Ser Thr Thr Val
Leu Cys 485 490 495 Gln Lys Leu Met Gln Val Val Pro Gln Glu Thr 500
505 <210> SEQ ID NO 3 <211> LENGTH: 339 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: STEAP1 <400> SEQUENCE: 3 Met
Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met 1 5 10
15 Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr
20 25 30 Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu
His Gln 35 40 45 Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu
Leu Gln His Thr 50 55 60 Gln Glu Leu Phe Pro Gln Trp His Leu Pro
Ile Lys Ile Ala Ala Ile 65 70 75 80 Ile Ala Ser Leu Thr Phe Leu Tyr
Thr Leu Leu Arg Glu Val Ile His 85 90 95 Pro Leu Ala Thr Ser His
Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu 100 105 110 Val Ile Asn Lys
Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125 Val Tyr
Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140
Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr 145
150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu
His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr
Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln
Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp Arg Met
Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala Ile Leu
Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val Ser Asp
Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys 245 250 255 Leu
Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His Ala Leu Ile Phe 260 265
270 Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro
275 280 285 Pro Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val Val Leu
Ile Phe 290 295 300 Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys
Ile Leu Lys Ile 305 310 315 320 Arg His Gly Trp Glu Asp Val Thr Lys
Ile Asn Lys Thr Glu Ile Cys 325 330 335 Ser Gln Leu <210> SEQ
ID NO 4 <211> LENGTH: 6995 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: MUC16 <400> SEQUENCE: 4 Pro Val Thr Ser Leu Leu
Thr Pro Gly Leu Val Ile Thr Thr Asp Arg 1 5 10 15 Met Gly Ile Ser
Arg Glu Pro Gly Thr Ser Ser Thr Ser Asn Leu Ser 20 25 30 Ser Thr
Ser His Glu Arg Leu Thr Thr Leu Glu Asp Thr Val Asp Thr 35 40 45
Glu Ala Met Gln Pro Ser Thr His Thr Ala Val Thr Asn Val Arg Thr 50
55 60 Ser Ile Ser Gly His Glu Ser Gln Ser Ser Val Leu Ser Asp Ser
Glu 65 70 75 80 Thr Pro Lys Ala Thr Ser Pro Met Gly Thr Thr Tyr Thr
Met Gly Glu 85 90 95 Thr Ser Val Ser Ile Ser Thr Ser Asp Phe Phe
Glu Thr Ser Arg Ile 100 105 110 Gln Ile Glu Pro Thr Ser Ser Leu Thr
Ser Gly Leu Arg Glu Thr Ser 115 120 125 Ser Ser Glu Arg Ile Ser Ser
Ala Thr Glu Gly Ser Thr Val Leu Ser 130 135 140 Glu Val Pro Ser Gly
Ala Thr Thr Glu Val Ser Arg Thr Glu Val Ile 145 150 155 160 Ser Ser
Arg Gly Thr Ser Met Ser Gly Pro Asp Gln Phe Thr Ile Ser 165 170 175
Pro Asp Ile Ser Thr Glu Ala Ile Thr Arg Leu Ser Thr Ser Pro Ile 180
185 190 Met Thr Glu Ser Ala Glu Ser Ala Ile Thr Ile Glu Thr Gly Ser
Pro 195 200 205 Gly Ala Thr Ser Glu Gly Thr Leu Thr Leu Asp Thr Ser
Thr Thr Thr 210 215 220 Phe Trp Ser Gly Thr His Ser Thr Ala Ser Pro
Gly Phe Ser His Ser 225 230 235 240 Glu Met Thr Thr Leu Met Ser Arg
Thr Pro Gly Asp Val Pro Trp Pro 245 250 255 Ser Leu Pro Ser Val Glu
Glu Ala Ser Ser Val Ser Ser Ser Leu Ser 260 265 270 Ser Pro Ala Met
Thr Ser Thr Ser Phe Phe Ser Thr Leu Pro Glu Ser 275 280 285 Ile Ser
Ser Ser Pro His Pro Val Thr Ala Leu Leu Thr Leu Gly Pro 290 295 300
Val Lys Thr Thr Asp Met Leu Arg Thr Ser Ser Glu Pro Glu Thr Ser 305
310 315 320 Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu Ile Leu Ala
Thr Ser 325 330 335 Glu Val Thr Lys Asp Arg Glu Lys Ile His Pro Ser
Ser Asn Thr Pro 340 345 350 Val Val Asn Val Gly Thr Val Ile Tyr Lys
His Leu Ser Pro Ser Ser 355 360 365 Val Leu Ala Asp Leu Val Thr Thr
Lys Pro Thr Ser Pro Met Ala Thr 370 375 380 Thr Ser Thr Leu Gly Asn
Thr Ser Val Ser Thr Ser Thr Pro Ala Phe 385 390 395 400 Pro Glu Thr
Met Met Thr Gln Pro Thr Ser Ser Leu Thr Ser Gly Leu 405 410 415 Arg
Glu Ile Ser Thr Ser Gln Glu Thr Ser Ser Ala Thr Glu Arg Ser 420 425
430 Ala Ser Leu Ser Gly Met Pro Thr Gly Ala Thr Thr Lys Val Ser Arg
435 440 445 Thr Glu Ala Leu Ser Leu Gly Arg Thr Ser Thr Pro Gly Pro
Ala Gln 450 455 460 Ser Thr Ile Ser Pro Glu Ile Ser Thr Glu Thr Ile
Thr Arg Ile Ser 465 470 475 480 Thr Pro Leu Thr Thr Thr Gly Ser Ala
Glu Met Thr Ile Thr Pro Lys 485 490 495 Thr Gly His Ser Gly Ala Ser
Ser Gln Gly Thr Phe Thr Leu Asp Thr 500 505 510 Ser Ser Arg Ala Ser
Trp Pro Gly Thr His Ser Ala Ala Thr His Arg 515 520 525 Ser Pro His
Ser Gly Met Thr Thr Pro Met Ser Arg Gly Pro Glu Asp 530 535 540 Val
Ser Trp Pro Ser Arg Pro Ser Val Glu Lys Thr Ser Pro Pro Ser 545 550
555 560 Ser Leu Val Ser Leu Ser Ala Val Thr Ser Pro Ser Pro Leu Tyr
Ser 565 570 575 Thr Pro Ser Glu Ser Ser His Ser Ser Pro Leu Arg Val
Thr Ser Leu 580 585 590 Phe Thr Pro Val Met Met Lys Thr Thr Asp Met
Leu Asp Thr Ser Leu 595 600 605 Glu Pro Val Thr Thr Ser Pro Pro Ser
Met Asn Ile Thr Ser Asp Glu 610 615 620 Ser Leu Ala Thr Ser Lys Ala
Thr Met Glu Thr Glu Ala Ile Gln Leu 625 630 635 640 Ser Glu Asn Thr
Ala Val Thr Gln Met Gly Thr Ile Ser Ala Arg Gln 645 650 655 Glu Phe
Tyr Ser Ser Tyr Pro Gly Leu Pro Glu Pro Ser Lys Val Thr 660 665 670
Ser Pro Val Val Thr Ser Ser Thr Ile Lys Asp Ile Val Ser Thr Thr 675
680 685 Ile Pro Ala Ser Ser Glu Ile Thr Arg Ile Glu Met Glu Ser Thr
Ser 690 695 700 Thr Leu Thr Pro Thr Pro Arg Glu Thr Ser Thr Ser Gln
Glu Ile His 705 710 715 720 Ser Ala Thr Lys Pro Ser Thr Val Pro Tyr
Lys Ala Leu Thr Ser Ala 725 730 735 Thr Ile Glu Asp Ser Met Thr Gln
Val Met Ser Ser Ser Arg Gly Pro 740 745 750 Ser Pro Asp Gln Ser Thr
Met Ser Gln Asp Ile Ser Thr Glu Val Ile 755 760 765 Thr Arg Leu Ser
Thr Ser Pro Ile Lys Thr Glu Ser Thr Glu Met Thr 770 775 780 Ile Thr
Thr Gln Thr Gly Ser Pro Gly Ala Thr Ser Arg Gly Thr Leu 785 790 795
800 Thr Leu Asp Thr Ser Thr Thr Phe Met Ser Gly Thr His Ser Thr Ala
805 810 815 Ser Gln Gly Phe Ser His Ser Gln Met Thr Ala Leu Met Ser
Arg Thr 820 825 830 Pro Gly Glu Val Pro Trp Leu Ser His Pro Ser Val
Glu Glu Ala Ser 835 840 845 Ser Ala Ser Phe Ser Leu Ser Ser Pro Val
Met Thr Ser Ser Ser Pro 850 855 860 Val Ser Ser Thr Leu Pro Asp Ser
Ile His Ser Ser Ser Leu Pro Val 865 870 875 880 Thr Ser Leu Leu Thr
Ser Gly Leu Val Lys Thr Thr Glu Leu Leu Gly 885 890 895 Thr Ser Ser
Glu Pro Glu Thr Ser Ser Pro Pro Asn Leu Ser Ser Thr 900 905 910 Ser
Ala Glu Ile Leu Ala Thr Thr Glu Val Thr Thr Asp Thr Glu Lys 915 920
925 Leu Glu Met Thr Asn Val Val Thr Ser Gly Tyr Thr His Glu Ser Pro
930 935 940 Ser Ser Val Leu Ala Asp Ser Val Thr Thr Lys Ala Thr Ser
Ser Met 945 950 955 960 Gly Ile Thr Tyr Pro Thr Gly Asp Thr Asn Val
Leu Thr Ser Thr Pro 965 970 975 Ala Phe Ser Asp Thr Ser Arg Ile Gln
Thr Lys Ser Lys Leu Ser Leu 980 985 990 Thr Pro Gly Leu Met Glu Thr
Ser Ile Ser Glu Glu Thr Ser Ser Ala 995 1000 1005 Thr Glu Lys Ser
Thr Val Leu Ser Ser Val Pro Thr Gly Ala Thr Thr 1010 1015 1020 Glu
Val Ser Arg Thr Glu Ala Ile Ser Ser Ser Arg Thr Ser Ile Pro 1025
1030 1035 1040 Gly Pro Ala Gln Ser Thr Met Ser Ser Asp Thr Ser Met
Glu Thr Ile 1045 1050 1055 Thr Arg Ile Ser Thr Pro Leu Thr Arg Lys
Glu Ser Thr Asp Met Ala 1060 1065 1070 Ile Thr Pro Lys Thr Gly Pro
Ser Gly Ala Thr Ser Gln Gly Thr Phe 1075 1080 1085 Thr Leu Asp Ser
Ser Ser Thr Ala Ser Trp Pro Gly Thr His Ser Ala 1090 1095 1100 Thr
Thr Gln Arg Phe Pro Arg Ser Val Val Thr Thr Pro Met Ser Arg 1105
1110 1115 1120 Gly Pro Glu Asp Val Ser Trp Pro Ser Pro Leu Ser Val
Glu Lys Asn 1125 1130 1135 Ser Pro Pro Ser Ser Leu Val Ser Ser Ser
Ser Val Thr Ser Pro Ser 1140 1145 1150 Pro Leu Tyr Ser Thr Pro Ser
Gly Ser Ser His Ser Ser Pro Val Pro 1155 1160 1165 Val Thr Ser Leu
Phe Thr Ser Ile Met Met Lys Ala Thr Asp Met Leu 1170 1175 1180 Asp
Ala Ser Leu Glu Pro Glu Thr Thr Ser Ala Pro Asn Met Asn Ile 1185
1190 1195 1200 Thr Ser Asp Glu Ser Leu Ala Ala Ser Lys Ala Thr Thr
Glu Thr Glu 1205 1210 1215 Ala Ile His Val Phe Glu Asn Thr Ala Ala
Ser His Val Glu Thr Thr 1220 1225 1230 Ser Ala Thr Glu Glu Leu Tyr
Ser Ser Ser Pro Gly Phe Ser Glu Pro 1235 1240 1245 Thr Lys Val Ile
Ser Pro Val Val Thr Ser Ser Ser Ile Arg Asp Asn 1250 1255 1260 Met
Val Ser Thr Thr Met Pro Gly Ser Ser Gly Ile Thr Arg Ile Glu 1265
1270 1275 1280 Ile Glu Ser Met Ser Ser Leu Thr Pro Gly Leu Arg Glu
Thr Arg Thr 1285 1290 1295 Ser Gln Asp Ile Thr Ser Ser Thr Glu Thr
Ser Thr Val Leu Tyr Lys 1300 1305 1310 Met Pro Ser Gly Ala Thr Pro
Glu Val Ser Arg Thr Glu Val Met Pro 1315 1320 1325 Ser Ser Arg Thr
Ser Ile Pro Gly Pro Ala Gln Ser Thr Met Ser Leu 1330 1335 1340 Asp
Ile Ser Asp Glu Val Val Thr Arg Leu Ser Thr Ser Pro Ile Met 1345
1350 1355 1360 Thr Glu Ser Ala Glu Ile Thr Ile Thr Thr Gln Thr Gly
Tyr Ser Leu 1365 1370 1375 Ala Thr Ser Gln Val Thr Leu Pro Leu Gly
Thr Ser Met Thr Phe Leu 1380 1385 1390 Ser Gly Thr His Ser Thr Met
Ser Gln Gly Leu Ser His Ser Glu Met 1395 1400 1405 Thr Asn Leu Met
Ser Arg Gly Pro Glu Ser Leu Ser Trp Thr Ser Pro 1410 1415 1420 Arg
Phe Val Glu Thr Thr Arg Ser Ser Ser Ser Leu Thr Ser Leu Pro 1425
1430 1435 1440 Leu Thr Thr Ser Leu Ser Pro Val Ser Ser Thr Leu Leu
Asp Ser Ser 1445 1450 1455 Pro Ser Ser Pro Leu Pro Val Thr Ser Leu
Ile Leu Pro Gly Leu Val 1460 1465 1470 Lys Thr Thr Glu Val Leu Asp
Thr Ser Ser Glu Pro Lys Thr Ser Ser 1475 1480 1485 Ser Pro Asn Leu
Ser Ser Thr Ser Val Glu Ile Pro Ala Thr Ser Glu 1490 1495 1500 Ile
Met Thr Asp Thr Glu Lys Ile His Pro Ser Ser Asn Thr Ala Val 1505
1510 1515 1520 Ala Lys Val Arg Thr Ser Ser Ser Val His Glu Ser His
Ser Ser Val 1525 1530 1535 Leu Ala Asp Ser Glu Thr Thr Ile Thr Ile
Pro Ser Met Gly Ile Thr 1540 1545 1550 Ser Ala Val Glu Asp Thr Thr
Val Phe Thr Ser Asn Pro Ala Phe Ser 1555 1560 1565 Glu Thr Arg Arg
Ile Pro Thr Glu Pro Thr Phe Ser Leu Thr Pro Gly 1570 1575 1580 Phe
Arg Glu Thr Ser Thr Ser Glu Glu Thr Thr Ser Ile Thr Glu Thr 1585
1590 1595 1600 Ser Ala Val Leu Phe Gly Val Pro Thr Ser Ala Thr Thr
Glu Val Ser 1605 1610 1615 Met Thr Glu Ile Met Ser Ser Asn Arg Thr
His Ile Pro Asp Ser Asp 1620 1625 1630 Gln Ser Thr Met Ser Pro Asp
Ile Ile Thr Glu Val Ile Thr Arg Leu 1635 1640 1645 Ser Ser Ser Ser
Met Met Ser Glu Ser Thr Gln Met Thr Ile Thr Thr 1650 1655 1660 Gln
Lys Ser Ser Pro Gly Ala Thr Ala Gln Ser Thr Leu Thr Leu Ala 1665
1670 1675 1680 Thr Thr Thr Ala Pro Leu Ala Arg Thr His Ser Thr Val
Pro Pro Arg 1685 1690 1695 Phe Leu His Ser Glu Met Thr Thr Leu Met
Ser Arg Ser Pro Glu Asn 1700 1705 1710 Pro Ser Trp Lys Ser Ser Pro
Phe Val Glu Lys Thr Ser Ser Ser Ser 1715 1720 1725 Ser Leu Leu Ser
Leu Pro Val Thr Thr Ser Pro Ser Val Ser Ser Thr 1730 1735 1740 Leu
Pro Gln Ser Ile Pro Ser Ser Ser Phe Ser Val Thr Ser Leu Leu 1745
1750 1755 1760 Thr Pro Gly Met Val Lys Thr Thr Asp Thr Ser Thr Glu
Pro Gly Thr 1765 1770 1775 Ser Leu Ser Pro Asn Leu Ser Gly Thr Ser
Val Glu Ile Leu Ala Ala 1780 1785 1790 Ser Glu Val Thr Thr Asp Thr
Glu Lys Ile His Pro Ser Ser Ser Met 1795 1800 1805 Ala Val Thr Asn
Val Gly Thr Thr Ser Ser Gly His Glu Leu Tyr Ser 1810 1815 1820 Ser
Val Ser Ile His Ser Glu Pro Ser Lys Ala Thr Tyr Pro Val Gly 1825
1830 1835 1840 Thr Pro Ser Ser Met Ala Glu Thr Ser Ile Ser Thr Ser
Met Pro Ala 1845 1850 1855 Asn Phe Glu Thr Thr Gly Phe Glu Ala Glu
Pro Phe Ser His Leu Thr 1860 1865 1870 Ser Gly Leu Arg Lys Thr Asn
Met Ser Leu Asp Thr Ser Ser Val Thr 1875 1880 1885 Pro Thr Asn Thr
Pro Ser Ser Pro Gly Ser Thr His Leu Leu Gln Ser 1890 1895 1900 Ser
Lys Thr Asp Phe Thr Ser Ser Ala Lys Thr Ser Ser Pro Asp Trp 1905
1910 1915 1920 Pro Pro Ala Ser Gln Tyr Thr Glu Ile Pro Val Asp Ile
Ile Thr Pro 1925 1930 1935 Phe Asn Ala Ser Pro Ser Ile Thr Glu Ser
Thr Gly Ile Thr Ser Phe 1940 1945 1950 Pro Glu Ser Arg Phe Thr Met
Ser Val Thr Glu Ser Thr His His Leu 1955 1960 1965 Ser Thr Asp Leu
Leu Pro Ser Ala Glu Thr Ile Ser Thr Gly Thr Val 1970 1975 1980 Met
Pro Ser Leu Ser Glu Ala Met Thr Ser Phe Ala Thr Thr Gly Val 1985
1990 1995 2000 Pro Arg Ala Ile Ser Gly Ser Gly Ser Pro Phe Ser Arg
Thr Glu Ser 2005 2010 2015 Gly Pro Gly Asp Ala Thr Leu Ser Thr Ile
Ala Glu Ser Leu Pro Ser 2020 2025 2030 Ser Thr Pro Val Pro Phe Ser
Ser Ser Thr Phe Thr Thr Thr Asp Ser 2035 2040 2045 Ser Thr Ile Pro
Ala Leu His Glu Ile Thr Ser Ser Ser Ala Thr Pro 2050 2055 2060 Tyr
Arg Val Asp Thr Ser Leu Gly Thr Glu Ser Ser Thr Thr Glu Gly 2065
2070 2075 2080 Arg Leu Val Met Val Ser Thr Leu Asp Thr Ser Ser Gln
Pro Gly Arg 2085 2090 2095 Thr Ser Ser Ser Pro Ile Leu Asp Thr Arg
Met Thr Glu Ser Val Glu 2100 2105 2110 Leu Gly Thr Val Thr Ser Ala
Tyr Gln Val Pro Ser Leu Ser Thr Arg 2115 2120 2125 Leu Thr Arg Thr
Asp Gly Ile Met Glu His Ile Thr Lys Ile Pro Asn 2130 2135 2140 Glu
Ala Ala His Arg Gly Thr Ile Arg Pro Val Lys Gly Pro Gln Thr 2145
2150 2155 2160 Ser Thr Ser Pro Ala Ser Pro Lys Gly Leu His Thr Gly
Gly Thr Lys 2165 2170 2175 Arg Met Glu Thr Thr Thr Thr Ala Leu Lys
Thr Thr Thr Thr Ala Leu 2180 2185 2190 Lys Thr Thr Ser Arg Ala Thr
Leu Thr Thr Ser Val Tyr Thr Pro Thr 2195 2200 2205 Leu Gly Thr Leu
Thr Pro Leu Asn Ala Ser Met Gln Met Ala Ser Thr 2210 2215 2220 Ile
Pro Thr Glu Met Met Ile Thr Thr Pro Tyr Val Phe Pro Asp Val 2225
2230 2235 2240 Pro Glu Thr Thr Ser Ser Leu Ala Thr Ser Leu Gly Ala
Glu Thr Ser 2245 2250 2255 Thr Ala Leu Pro Arg Thr Thr Pro Ser Val
Phe Asn Arg Glu Ser Glu 2260 2265 2270 Thr Thr Ala Ser Leu Val Ser
Arg Ser Gly Ala Glu Arg Ser Pro Val 2275 2280 2285 Ile Gln Thr Leu
Asp Val Ser Ser Ser Glu Pro Asp Thr Thr Ala Ser 2290 2295 2300 Trp
Val Ile His Pro Ala Glu Thr Ile Pro Thr Val Ser Lys Thr Thr 2305
2310 2315 2320 Pro Asn Phe Phe His Ser Glu Leu Asp Thr Val Ser Ser
Thr Ala Thr 2325 2330 2335 Ser His Gly Ala Asp Val Ser Ser Ala Ile
Pro Thr Asn Ile Ser Pro 2340 2345 2350 Ser Glu Leu Asp Ala Leu Thr
Pro Leu Val Thr Ile Ser Gly Thr Asp 2355 2360 2365 Thr Ser Thr Thr
Phe Pro Thr Leu Thr Lys Ser Pro His Glu Thr Glu 2370 2375 2380 Thr
Arg Thr Thr Trp Leu Thr His Pro Ala Glu Thr Ser Ser Thr Ile 2385
2390 2395 2400 Pro Arg Thr Ile Pro Asn Phe Ser His His Glu Ser Asp
Ala Thr Pro 2405 2410 2415 Ser Ile Ala Thr Ser Pro Gly Ala Glu Thr
Ser Ser Ala Ile Pro Ile 2420 2425 2430 Met Thr Val Ser Pro Gly Ala
Glu Asp Leu Val Thr Ser Gln Val Thr 2435 2440 2445 Ser Ser Gly Thr
Asp Arg Asn Met Thr Ile Pro Thr Leu Thr Leu Ser 2450 2455 2460 Pro
Gly Glu Pro Lys Thr Ile Ala Ser Leu Val Thr His Pro Glu Ala 2465
2470 2475 2480 Gln Thr Ser Ser Ala Ile Pro Thr Ser Thr Ile Ser Pro
Ala Val Ser 2485 2490 2495 Arg Leu Val Thr Ser Met Val Thr Ser Leu
Ala Ala Lys Thr Ser Thr 2500 2505 2510 Thr Asn Arg Ala Leu Thr Asn
Ser Pro Gly Glu Pro Ala Thr Thr Val 2515 2520 2525 Ser Leu Val Thr
His Ser Ala Gln Thr Ser Pro Thr Val Pro Trp Thr 2530 2535 2540 Thr
Ser Ile Phe Phe His Ser Lys Ser Asp Thr Thr Pro Ser Met Thr 2545
2550 2555 2560 Thr Ser His Gly Ala Glu Ser Ser Ser Ala Val Pro Thr
Pro Thr Val 2565 2570 2575 Ser Thr Glu Val Pro Gly Val Val Thr Pro
Leu Val Thr Ser Ser Arg 2580 2585 2590 Ala Val Ile Ser Thr Thr Ile
Pro Ile Leu Thr Leu Ser Pro Gly Glu 2595 2600 2605 Pro Glu Thr Thr
Pro Ser Met Ala Thr Ser His Gly Glu Glu Ala Ser 2610 2615 2620 Ser
Ala Ile Pro Thr Pro Thr Val Ser Pro Gly Val Pro Gly Val Val 2625
2630 2635 2640 Thr Ser Leu Val Thr Ser Ser Arg Ala Val Thr Ser Thr
Thr Ile Pro 2645 2650 2655 Ile Leu Thr Phe Ser Leu Gly Glu Pro Glu
Thr Thr Pro Ser Met Ala 2660 2665 2670 Thr Ser His Gly Thr Glu Ala
Gly Ser Ala Val Pro Thr Val Leu Pro 2675 2680 2685 Glu Val Pro Gly
Met Val Thr Ser Leu Val Ala Ser Ser Arg Ala Val 2690 2695 2700 Thr
Ser Thr Thr Leu Pro Thr Leu Thr Leu Ser Pro Gly Glu Pro Glu 2705
2710 2715 2720 Thr Thr Pro Ser Met Ala Thr Ser His Gly Ala Glu Ala
Ser Ser Thr 2725 2730 2735 Val Pro Thr Val Ser Pro Glu Val Pro Gly
Val Val Thr Ser Leu Val 2740 2745 2750 Thr Ser Ser Ser Gly Val Asn
Ser Thr Ser Ile Pro Thr Leu Ile Leu 2755 2760 2765 Ser Pro Gly Glu
Leu Glu Thr Thr Pro Ser Met Ala Thr Ser His Gly 2770 2775 2780 Ala
Glu Ala Ser Ser Ala Val Pro Thr Pro Thr Val Ser Pro Gly Val 2785
2790 2795 2800 Ser Gly Val Val Thr Pro Leu Val Thr Ser Ser Arg Ala
Val Thr Ser 2805 2810 2815 Thr Thr Ile Pro Ile Leu Thr Leu Ser Ser
Ser Glu Pro Glu Thr Thr 2820 2825 2830 Pro Ser Met Ala Thr Ser His
Gly Val Glu Ala Ser Ser Ala Val Leu 2835 2840 2845 Thr Val Ser Pro
Glu Val Pro Gly Met Val Thr Phe Leu Val Thr Ser 2850 2855 2860 Ser
Arg Ala Val Thr Ser Thr Thr Ile Pro Thr Leu Thr Ile Ser Ser 2865
2870 2875 2880 Asp Glu Pro Glu Thr Thr Thr Ser Leu Val Thr His Ser
Glu Ala Lys 2885 2890 2895 Met Ile Ser Ala Ile Pro Thr Leu Gly Val
Ser Pro Thr Val Gln Gly 2900 2905 2910 Leu Val Thr Ser Leu Val Thr
Ser Ser Gly Ser Glu Thr Ser Ala Phe 2915 2920 2925 Ser Asn Leu Thr
Val Ala Ser Ser Gln Pro Glu Thr Ile Asp Ser Trp 2930 2935 2940 Val
Ala His Pro Gly Thr Glu Ala Ser Ser Val Val Pro Thr Leu Thr 2945
2950 2955 2960 Val Ser Thr Gly Glu Pro Phe Thr Asn Ile Ser Leu Val
Thr His Pro 2965 2970 2975 Ala Glu Ser Ser Ser Thr Leu Pro Arg Thr
Thr Ser Arg Phe Ser His 2980 2985 2990 Ser Glu Leu Asp Thr Met Pro
Ser Thr Val Thr Ser Pro Glu Ala Glu 2995 3000 3005 Ser Ser Ser Ala
Ile Ser Thr Thr Ile Ser Pro Gly Ile Pro Gly Val 3010 3015 3020 Leu
Thr Ser Leu Val Thr Ser Ser Gly Arg Asp Ile Ser Ala Thr Phe 3025
3030 3035 3040 Pro Thr Val Pro Glu Ser Pro His Glu Ser Glu Ala Thr
Ala Ser Trp 3045 3050 3055 Val Thr His Pro Ala Val Thr Ser Thr Thr
Val Pro Arg Thr Thr Pro 3060 3065 3070 Asn Tyr Ser His Ser Glu Pro
Asp Thr Thr Pro Ser Ile Ala Thr Ser 3075 3080 3085 Pro Gly Ala Glu
Ala Thr Ser Asp Phe Pro Thr Ile Thr Val Ser Pro 3090 3095 3100 Asp
Val Pro Asp Met Val Thr Ser Gln Val Thr Ser Ser Gly Thr Asp 3105
3110 3115 3120 Thr Ser Ile Thr Ile Pro Thr Leu Thr Leu Ser Ser Gly
Glu Pro Glu 3125 3130 3135 Thr Thr Thr Ser Phe Ile Thr Tyr Ser Glu
Thr His Thr Ser Ser Ala 3140 3145 3150 Ile Pro Thr Leu Pro Val Ser
Pro Asp Ala Ser Lys Met Leu Thr Ser 3155 3160 3165 Leu Val Ile Ser
Ser Gly Thr Asp Ser Thr Thr Thr Phe Pro Thr Leu 3170 3175 3180 Thr
Glu Thr Pro Tyr Glu Pro Glu Thr Thr Ala Ile Gln Leu Ile His 3185
3190 3195 3200 Pro Ala Glu Thr Asn Thr Met Val Pro Arg Thr Thr Pro
Lys Phe Ser 3205 3210 3215 His Ser Lys Ser Asp Thr Thr Leu Pro Val
Ala Ile Thr Ser Pro Gly 3220 3225 3230 Pro Glu Ala Ser Ser Ala Val
Ser Thr Thr Thr Ile Ser Pro Asp Met 3235 3240 3245 Ser Asp Leu Val
Thr Ser Leu Val Pro Ser Ser Gly Thr Asp Thr Ser 3250 3255 3260 Thr
Thr Phe Pro Thr Leu Ser Glu Thr Pro Tyr Glu Pro Glu Thr Thr 3265
3270 3275 3280 Ala Thr Trp Leu Thr His Pro Ala Glu Thr Ser Thr Thr
Val Ser Gly 3285 3290 3295 Thr Ile Pro Asn Phe Ser His Arg Gly Ser
Asp Thr Ala Pro Ser Met 3300 3305 3310 Val Thr Ser Pro Gly Val Asp
Thr Arg Ser Gly Val Pro Thr Thr Thr 3315 3320 3325 Ile Pro Pro Ser
Ile Pro Gly Val Val Thr Ser Gln Val Thr Ser Ser 3330 3335 3340 Ala
Thr Asp Thr Ser Thr Ala Ile Pro Thr Leu Thr Pro Ser Pro Gly 3345
3350 3355 3360 Glu Pro Glu Thr Thr Ala Ser Ser Ala Thr His Pro Gly
Thr Gln Thr 3365 3370 3375 Gly Phe Thr Val Pro Ile Arg Thr Val Pro
Ser Ser Glu Pro Asp Thr 3380 3385 3390 Met Ala Ser Trp Val Thr His
Pro Pro Gln Thr Ser Thr Pro Val Ser 3395 3400 3405 Arg Thr Thr Ser
Ser Phe Ser His Ser Ser Pro Asp Ala Thr Pro Val 3410 3415 3420 Met
Ala Thr Ser Pro Arg Thr Glu Ala Ser Ser Ala Val Leu Thr Thr 3425
3430 3435 3440 Ile Ser Pro Gly Ala Pro Glu Met Val Thr Ser Gln Ile
Thr Ser Ser 3445 3450 3455 Gly Ala Ala Thr Ser Thr Thr Val Pro Thr
Leu Thr His Ser Pro Gly 3460 3465 3470 Met Pro Glu Thr Thr Ala Leu
Leu Ser Thr His Pro Arg Thr Glu Thr 3475 3480 3485 Ser Lys Thr Phe
Pro Ala Ser Thr Val Phe Pro Gln Val Ser Glu Thr 3490 3495 3500 Thr
Ala Ser Leu Thr Ile Arg Pro Gly Ala Glu Thr Ser Thr Ala Leu 3505
3510 3515 3520 Pro Thr Gln Thr Thr Ser Ser Leu Phe Thr Leu Leu Val
Thr Gly Thr 3525 3530 3535 Ser Arg Val Asp Leu Ser Pro Thr Ala Ser
Pro Gly Val Ser Ala Lys 3540 3545 3550 Thr Ala Pro Leu Ser Thr His
Pro Gly Thr Glu Thr Ser Thr Met Ile 3555 3560 3565 Pro Thr Ser Thr
Leu Ser Leu Gly Leu Leu Glu Thr Thr Gly Leu Leu 3570 3575 3580 Ala
Thr Ser Ser Ser Ala Glu Thr Ser Thr Ser Thr Leu Thr Leu Thr 3585
3590 3595 3600 Val Ser Pro Ala Val Ser Gly Leu Ser Ser Ala Ser Ile
Thr Thr Asp 3605 3610 3615 Lys Pro Gln Thr Val Thr Ser Trp Asn Thr
Glu Thr Ser Pro Ser Val 3620 3625 3630 Thr Ser Val Gly Pro Pro Glu
Phe Ser Arg Thr Val Thr Gly Thr Thr 3635 3640 3645 Met Thr Leu Ile
Pro Ser Glu Met Pro Thr Pro Pro Lys Thr Ser His 3650 3655 3660 Gly
Glu Gly Val Ser Pro Thr Thr Ile Leu Arg Thr Thr Met Val Glu 3665
3670 3675 3680 Ala Thr Asn Leu Ala Thr Thr Gly Ser Ser Pro Thr Val
Ala Lys Thr 3685 3690 3695 Thr Thr Thr Phe Asn Thr Leu Ala Gly Ser
Leu Phe Thr Pro Leu Thr 3700 3705 3710 Thr Pro Gly Met Ser Thr Leu
Ala Ser Glu Ser Val Thr Ser Arg Thr 3715 3720 3725 Ser Tyr Asn His
Arg Ser Trp Ile Ser Thr Thr Ser Ser Tyr Asn Arg 3730 3735 3740 Arg
Tyr Trp Thr Pro Ala Thr Ser Thr Pro Val Thr Ser Thr Phe Ser 3745
3750 3755 3760 Pro Gly Ile Ser Thr Ser Ser Ile Pro Ser Ser Thr Ala
Ala Thr Val 3765 3770 3775 Pro Phe Met Val Pro Phe Thr Leu Asn Phe
Thr Ile Thr Asn Leu Gln 3780 3785 3790 Tyr Glu Glu Asp Met Arg His
Pro Gly Ser Arg Lys Phe Asn Ala Thr 3795 3800 3805 Glu Arg Glu Leu
Gln Gly Leu Leu Lys Pro Leu Phe Arg Asn Ser Ser 3810 3815 3820 Leu
Glu Tyr Leu Tyr Ser Gly Cys Arg Leu Ala Ser Leu Arg Pro Glu 3825
3830 3835 3840 Lys Asp Ser Ser Ala Thr Ala Val Asp Ala Ile Cys Thr
His Arg Pro 3845 3850 3855 Asp Pro Glu Asp Leu Gly Leu Asp Arg Glu
Arg Leu Tyr Trp Glu Leu 3860 3865 3870 Ser Asn Leu Thr Asn Gly Ile
Gln Glu Leu Gly Pro Tyr Thr Leu Asp 3875 3880 3885 Arg Asn Ser Leu
Tyr Val Asn Gly Phe Thr His Arg Ser Ser Met Pro 3890 3895 3900 Thr
Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Val Gly Thr Ser Gly 3905
3910 3915 3920 Thr Pro Ser Ser Ser Pro Ser Pro Thr Thr Ala Gly Pro
Leu Leu Met 3925 3930 3935 Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn
Leu Gln Tyr Glu Glu Asp 3940 3945 3950 Met Arg Arg Thr Gly Ser Arg
Lys Phe Asn Thr Met Glu Ser Val Leu 3955 3960 3965 Gln Gly Leu Leu
Lys Pro Leu Phe Lys Asn Thr Ser Val Gly Pro Leu 3970 3975 3980 Tyr
Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Ala 3985
3990 3995 4000 Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Leu Asp
Pro Lys Ser 4005 4010 4015 Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp
Glu Leu Ser Lys Leu Thr 4020 4025 4030 Asn Asp Ile Glu Glu Leu Gly
Pro Tyr Thr Leu Asp Arg Asn Ser Leu 4035 4040 4045 Tyr Val Asn Gly
Phe Thr His Gln Ser Ser Val Ser Thr Thr Ser Thr 4050 4055 4060 Pro
Gly Thr Ser Thr Val Asp Leu Arg Thr Ser Gly Thr Pro Ser Ser 4065
4070 4075 4080 Leu Ser Ser Pro Thr Ile Met Ala Ala Gly Pro Leu Leu
Val Pro Phe 4085 4090 4095 Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln
Tyr Gly Glu Asp Met Gly 4100 4105 4110 His Pro Gly Ser Arg Lys Phe
Asn Thr Thr Glu Arg Val Leu Gln Gly 4115 4120 4125 Leu Leu Gly Pro
Ile Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser 4130 4135 4140 Gly
Cys Arg Leu Thr Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr 4145
4150 4155 4160 Gly Val Asp Ala Ile Cys Ile His His Leu Asp Pro Lys
Ser Pro Gly 4165 4170 4175 Leu Asn Arg Glu Arg Leu Tyr Trp Glu Leu
Ser Gln Leu Thr Asn Gly 4180 4185 4190 Ile Lys Glu Leu Gly Pro Tyr
Thr Leu Asp Arg Asn Ser Leu Tyr Val 4195 4200 4205 Asn Gly Phe Thr
His Arg Thr Ser Val Pro Thr Thr Ser Thr Pro Gly 4210 4215 4220 Thr
Ser Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Phe Ser Leu Pro 4225
4230 4235 4240 Ser Pro Ala Thr Ala Gly Pro Leu Leu Val Leu Phe Thr
Leu Asn Phe 4245 4250 4255 Thr Ile Thr Asn Leu Lys Tyr Glu Glu Asp
Met His Arg Pro Gly Ser 4260 4265 4270 Arg Lys Phe Asn Thr Thr Glu
Arg Val Leu Gln Thr Leu Val Gly Pro 4275 4280 4285 Met Phe Lys Asn
Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu 4290 4295 4300 Thr
Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala 4305
4310 4315 4320 Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Val
Asp Arg Glu 4325 4330 4335 Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr
Asn Gly Ile Lys Glu Leu 4340 4345 4350 Gly Pro Tyr Thr Leu Asp Arg
Asn Ser Leu Tyr Val Asn Gly Phe Thr 4355 4360 4365 His Trp Ile Pro
Val Pro Thr Ser Ser Thr Pro Gly Thr Ser Thr Val 4370 4375 4380 Asp
Leu Gly Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala 4385
4390 4395 4400 Thr Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe
Thr Ile Thr 4405 4410 4415 Asn Leu Lys Tyr Glu Glu Asp Met His Cys
Pro Gly Ser Arg Lys Phe 4420 4425 4430 Asn Thr Thr Glu Arg Val Leu
Gln Ser Leu Leu Gly Pro Met Phe Lys 4435 4440 4445 Asn Thr Ser Val
Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu 4450 4455 4460 Arg
Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr 4465
4470 4475 4480 His Arg Leu Asp Pro Lys Ser Pro Gly Val Asp Arg Glu
Gln Leu Tyr 4485 4490 4495 Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile
Lys Glu Leu Gly Pro Tyr 4500 4505 4510 Thr Leu Asp Arg Asn Ser Leu
Tyr Val Asn Gly Phe Thr His Gln Thr 4515 4520 4525 Ser Ala Pro Asn
Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly 4530 4535 4540 Thr
Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala Gly Pro 4545
4550 4555 4560 Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn
Leu Gln Tyr 4565 4570 4575 Glu Glu Asp Met His His Pro Gly Ser Arg
Lys Phe Asn Thr Thr Glu 4580 4585 4590 Arg Val Leu Gln Gly Leu Leu
Gly Pro Met Phe Lys Asn Thr Ser Val 4595 4600 4605 Gly Leu Leu Tyr
Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys 4610 4615 4620 Asn
Gly Ala Ala Thr Gly Met Asp Ala Ile Cys Ser His Arg Leu Asp 4625
4630 4635 4640 Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp
Glu Leu Ser 4645 4650 4655 Gln Leu Thr His Gly Ile Lys Glu Leu Gly
Pro Tyr Thr Leu Asp Arg 4660 4665 4670 Asn Ser Leu Tyr Val Asn Gly
Phe Thr His Arg Ser Ser Val Ala Pro 4675 4680 4685 Thr Ser Thr Pro
Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr 4690 4695 4700 Pro
Ser Ser Leu Pro Ser Pro Thr Thr Ala Val Pro Leu Leu Val Pro 4705
4710 4715 4720 Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Gly
Glu Asp Met 4725 4730 4735 Arg His Pro Gly Ser Arg Lys Phe Asn Thr
Thr Glu Arg Val Leu Gln 4740 4745 4750 Gly Leu Leu Gly Pro Leu Phe
Lys Asn Ser Ser Val Gly Pro Leu Tyr 4755 4760 4765 Ser Gly Cys Arg
Leu Ile Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala 4770 4775 4780 Thr
Gly Val Asp Ala Ile Cys Thr His His Leu Asn Pro Gln Ser Pro 4785
4790 4795 4800 Gly Leu Asp Arg Glu Gln Leu Tyr Trp Gln Leu Ser Gln
Met Thr Asn 4805 4810 4815 Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu
Asp Arg Asn Ser Leu Tyr 4820 4825 4830 Val Asn Gly Phe Thr His Arg
Ser Ser Gly Leu Thr Thr Ser Thr Pro 4835 4840 4845 Trp Thr Ser Thr
Val Asp Leu Gly Thr Ser Gly Thr Pro Ser Pro Val 4850 4855 4860 Pro
Ser Pro Thr Thr Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn 4865
4870 4875 4880 Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His
Arg Pro Gly 4885 4890 4895 Ser Arg Lys Phe Asn Ala Thr Glu Arg Val
Leu Gln Gly Leu Leu Ser 4900 4905 4910 Pro Ile Phe Lys Asn Ser Ser
Val Gly Pro Leu Tyr Ser Gly Cys Arg 4915 4920 4925 Leu Thr Ser Leu
Arg Pro Glu Lys Asp Gly Ala Ala Thr Gly Met Asp 4930 4935 4940 Ala
Val Cys Leu Tyr His Pro Asn Pro Lys Arg Pro Gly Leu Asp Arg 4945
4950 4955 4960 Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Asn
Ile Thr Glu 4965 4970 4975 Leu Gly Pro Tyr Ser Leu Asp Arg Asp Ser
Leu Tyr Val Asn Gly Phe 4980 4985 4990 Thr His Gln Asn Ser Val Pro
Thr Thr Ser Thr Pro Gly Thr Ser Thr 4995 5000 5005 Val Tyr Trp Ala
Thr Thr Gly Thr Pro Ser Ser Phe Pro Gly His Thr 5010 5015 5020 Glu
Pro Gly Pro Leu Leu Ile Pro Phe Thr Phe Asn Phe Thr Ile Thr 5025
5030 5035 5040 Asn Leu His Tyr Glu Glu Asn Met Gln His Pro Gly Ser
Arg Lys Phe 5045 5050 5055 Asn Thr Thr Glu Arg Val Leu Gln Gly Leu
Leu Lys Pro Leu Phe Lys 5060 5065 5070 Asn Thr Ser Val Gly Pro Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu 5075 5080 5085 Arg Pro Glu Lys
Gln Glu Ala Ala Thr Gly Val Asp Thr Ile Cys Thr 5090 5095 5100 His
Arg Val Asp Pro Ile Gly Pro Gly Leu Asp Arg Glu Arg Leu Tyr 5105
5110 5115 5120 Trp Glu Leu Ser Gln Leu Thr Asn Ser Ile Thr Glu Leu
Gly Pro Tyr 5125 5130 5135 Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn
Gly Phe Asn Pro Trp Ser 5140 5145 5150 Ser Val Pro Thr Thr Ser Thr
Pro Gly Thr Ser Thr Val His Leu Ala 5155 5160 5165 Thr Ser Gly Thr
Pro Ser Ser Leu Pro Gly His Thr Ala Pro Val Pro 5170 5175 5180 Leu
Leu Ile Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu His Tyr 5185
5190 5195 5200 Glu Glu Asn Met Gln His Pro Gly Ser Arg Lys Phe Asn
Thr Thr Glu 5205 5210 5215 Arg Val Leu Gln Gly Leu Leu Lys Pro Leu
Phe Lys Ser Thr Ser Val 5220 5225 5230 Gly Pro Leu Tyr Ser Gly Cys
Arg Leu Thr Leu Leu Arg Pro Glu Lys 5235 5240 5245 His Gly Ala Ala
Thr Gly Val Asp Ala Ile Cys Thr Leu Arg Leu Asp 5250 5255 5260 Pro
Thr Gly Pro Gly Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser 5265
5270 5275 5280 Gln Leu Thr Asn Ser Val Thr Glu Leu Gly Pro Tyr Thr
Leu Asp Arg 5285 5290 5295 Asp Ser Leu Tyr Val Asn Gly Phe Thr His
Arg Ser Ser Val Pro Thr 5300 5305 5310 Thr Ser Ile Pro Gly Thr Ser
Ala Val His Leu Glu Thr Ser Gly Thr 5315 5320 5325 Pro Ala Ser Leu
Pro Gly His Thr Ala Pro Gly Pro Leu Leu Val Pro 5330 5335 5340 Phe
Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met 5345
5350 5355 5360 Arg His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg
Val Leu Gln 5365 5370 5375 Gly Leu Leu Lys Pro Leu Phe Lys Ser Thr
Ser Val Gly Pro Leu Tyr 5380 5385 5390 Ser Gly Cys Arg Leu Thr Leu
Leu Arg Pro Glu Lys Arg Gly Ala Ala 5395 5400 5405 Thr Gly Val Asp
Thr Ile Cys Thr His Arg Leu Asp Pro Leu Asn Pro 5410 5415 5420 Gly
Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Lys Leu Thr Arg 5425
5430 5435 5440 Gly Ile Ile Glu Leu Gly Pro Tyr Leu Leu Asp Arg Gly
Ser Leu Tyr 5445 5450 5455 Val Asn Gly Phe Thr His Arg Asn Phe Val
Pro Ile Thr Ser Thr Pro 5460 5465 5470 Gly Thr Ser Thr Val His Leu
Gly Thr Ser Glu Thr Pro Ser Ser Leu 5475 5480 5485 Pro Arg Pro Ile
Val Pro Gly Pro Leu Leu Val Pro Phe Thr Leu Asn 5490 5495 5500 Phe
Thr Ile Thr Asn Leu Gln Tyr Glu Glu Ala Met Arg His Pro Gly 5505
5510 5515 5520 Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly
Leu Leu Arg 5525 5530 5535 Pro Leu Phe Lys Asn Thr Ser Ile Gly Pro
Leu Tyr Ser Ser Cys Arg 5540 5545 5550 Leu Thr Leu Leu Arg Pro Glu
Lys Asp Lys Ala Ala Thr Arg Val Asp 5555 5560 5565 Ala Ile Cys Thr
His His Pro Asp Pro Gln Ser Pro Gly Leu Asn Arg 5570 5575 5580 Glu
Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Gly Ile Thr Glu 5585
5590 5595 5600 Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val
Asp Gly Phe 5605 5610 5615 Thr His Trp Ser Pro Ile Pro Thr Thr Ser
Thr Pro Gly Thr Ser Ile 5620 5625 5630 Val Asn Leu Gly Thr Ser Gly
Ile Pro Pro Ser Leu Pro Glu Thr Thr 5635 5640 5645 Ala Thr Gly Pro
Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr 5650 5655 5660 Asn
Leu Gln Tyr Glu Glu Asn Met Gly His Pro Gly Ser Arg Lys Phe 5665
5670 5675 5680 Asn Ile Thr Glu Ser Val Leu Gln Gly Leu Leu Lys Pro
Leu Phe Lys 5685 5690 5695 Ser Thr Ser Val Gly Pro Leu Tyr Ser Gly
Cys Arg Leu Thr Leu Leu 5700 5705 5710 Arg Pro Glu Lys Asp Gly Val
Ala Thr Arg Val Asp Ala Ile Cys Thr 5715 5720 5725 His Arg Pro Asp
Pro Lys Ile Pro Gly Leu Asp Arg Gln Gln Leu Tyr 5730 5735 5740 Trp
Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly Pro Tyr 5745
5750 5755 5760 Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr
Gln Arg Ser 5765 5770 5775 Ser Val Pro Thr Thr Ser Thr Pro Gly Thr
Phe Thr Val Gln Pro Glu 5780 5785 5790 Thr Ser Glu Thr Pro Ser Ser
Leu Pro Gly Pro Thr Ala Thr Gly Pro 5795 5800 5805 Val Leu Leu Pro
Phe Thr Leu Asn Phe Thr Ile Ile Asn Leu Gln Tyr 5810 5815 5820 Glu
Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu 5825
5830 5835 5840 Arg Val Leu Gln Gly Leu Leu Met Pro Leu Phe Lys Asn
Thr Ser Val 5845 5850 5855 Ser Ser Leu Tyr Ser Gly Cys Arg Leu Thr
Leu Leu Arg Pro Glu Lys 5860 5865 5870 Asp Gly Ala Ala Thr Arg Val
Asp Ala Val Cys Thr His Arg Pro Asp 5875 5880 5885 Pro Lys Ser Pro
Gly Leu Asp Arg Glu Arg Leu Tyr Trp Lys Leu Ser 5890 5895 5900 Gln
Leu Thr His Gly Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg 5905
5910 5915 5920 His Ser Leu Tyr Val Asn Gly Phe Thr His Gln Ser Ser
Met Thr Thr 5925 5930 5935 Thr Arg Thr Pro Asp Thr Ser Thr Met His
Leu Ala Thr Ser Arg Thr 5940 5945 5950 Pro Ala Ser Leu Ser Gly Pro
Thr Thr Ala Ser Pro Leu Leu Val Leu 5955 5960 5965 Phe Thr Ile Asn
Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met 5970 5975 5980 His
His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln 5985
5990 5995 6000 Gly Leu Leu Arg Pro Val Phe Lys Asn Thr Ser Val Gly
Pro Leu Tyr 6005 6010 6015 Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro
Lys Lys Asp Gly Ala Ala 6020 6025 6030 Thr Lys Val Asp Ala Ile Cys
Thr Tyr Arg Pro Asp Pro Lys Ser Pro 6035 6040 6045 Gly Leu Asp Arg
Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His 6050 6055 6060 Ser
Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr 6065
6070 6075 6080 Val Asn Gly Phe Thr Gln Arg Ser Ser Val Pro Thr Thr
Ser Ile Pro 6085 6090 6095 Gly Thr Pro Thr Val Asp Leu Gly Thr Ser
Gly Thr Pro Val Ser Lys 6100 6105 6110 Pro Gly Pro Ser Ala Ala Ser
Pro Leu Leu Val Leu Phe Thr Leu Asn 6115 6120 6125 Phe Thr Ile Thr
Asn Leu Arg Tyr Glu Glu Asn Met Gln His Pro Gly 6130 6135 6140 Ser
Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg 6145
6150 6155 6160 Ser Leu Phe Lys Ser Thr Ser Val Gly Pro Leu Tyr Ser
Gly Cys Arg 6165 6170 6175 Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly
Thr Ala Thr Gly Val Asp 6180 6185 6190 Ala Ile Cys Thr His His Pro
Asp Pro Lys Ser Pro Arg Leu Asp Arg 6195 6200 6205 Glu Gln Leu Tyr
Trp Glu Leu Ser Gln Leu Thr His Asn Ile Thr Glu 6210 6215 6220 Leu
Gly Pro Tyr Ala Leu Asp Asn Asp Ser Leu Phe Val Asn Gly Phe 6225
6230 6235 6240 Thr His Arg Ser Ser Val Ser Thr Thr Ser Thr Pro Gly
Thr Pro Thr 6245 6250 6255 Val Tyr Leu Gly Ala Ser Lys Thr Pro Ala
Ser Ile Phe Gly Pro Ser 6260 6265 6270 Ala Ala Ser His Leu Leu Ile
Leu Phe Thr Leu Asn Phe Thr Ile Thr 6275 6280 6285 Asn Leu Arg Tyr
Glu Glu Asn Met Trp Pro Gly Ser Arg Lys Phe Asn 6290 6295 6300 Thr
Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe Lys Asn 6305
6310 6315 6320 Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr
Leu Leu Arg 6325 6330 6335 Pro Glu Lys Asp Gly Glu Ala Thr Gly Val
Asp Ala Ile Cys Thr His 6340 6345 6350 Arg Pro Asp Pro Thr Gly Pro
Gly Leu Asp Arg Glu Gln Leu Tyr Leu 6355 6360 6365 Glu Leu Ser Gln
Leu Thr His Ser Ile Thr Glu Leu Gly Pro Tyr Thr 6370 6375 6380 Leu
Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser 6385
6390 6395 6400 Val Pro Thr Thr Ser Thr Gly Val Val Ser Glu Glu Pro
Phe Thr Leu 6405 6410 6415 Asn Phe Thr Ile Asn Asn Leu Arg Tyr Met
Ala Asp Met Gly Gln Pro 6420 6425 6430 Gly Ser Leu Lys Phe Asn Ile
Thr Asp Asn Val Met Gln His Leu Leu 6435 6440 6445 Ser Pro Leu Phe
Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr Gly Cys 6450 6455 6460 Arg
Val Ile Ala Leu Arg Ser Val Lys Asn Gly Ala Glu Thr Arg Val 6465
6470 6475 6480 Asp Leu Leu Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro
Gly Leu Pro 6485 6490 6495 Ile Lys Gln Val Phe His Glu Leu Ser Gln
Gln Thr His Gly Ile Thr 6500 6505 6510 Arg Leu Gly Pro Tyr Ser Leu
Asp Lys Asp Ser Leu Tyr Leu Asn Gly 6515 6520 6525 Tyr Asn Glu Pro
Gly Pro Asp Glu Pro Pro Thr Thr Pro Lys Pro Ala 6530 6535 6540 Thr
Thr Phe Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala Met Gly Tyr 6545
6550 6555 6560 His Leu Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn
Leu Gln Tyr 6565 6570 6575 Ser Pro Asp Met Gly Lys Gly Ser Ala Thr
Phe Asn Ser Thr Glu Gly 6580 6585 6590 Val Leu Gln His Leu Leu Arg
Pro Leu Phe Gln Lys Ser Ser Met Gly 6595 6600 6605 Pro Phe Tyr Leu
Gly Cys Gln Leu Ile Ser Leu Arg Pro Glu Lys Asp 6610 6615 6620 Gly
Ala Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr His Pro Asp Pro 6625
6630 6635 6640 Val Gly Pro Gly Leu Asp Ile Gln Gln Leu Tyr Trp Glu
Leu Ser Gln 6645 6650 6655 Leu Thr His Gly Val Thr Gln Leu Gly Phe
Tyr Val Leu Asp Arg Asp 6660 6665 6670 Ser Leu Phe Ile Asn Gly Tyr
Ala Pro Gln Asn Leu Ser Ile Arg Gly 6675 6680 6685 Glu Tyr Gln Ile
Asn Phe His Ile Val Asn Trp Asn Leu Ser Asn Pro 6690 6695 6700 Asp
Pro Thr Ser Ser Glu Tyr Ile Thr Leu Leu Arg Asp Ile Gln Asp 6705
6710 6715 6720 Lys Val Thr Thr Leu Tyr Lys Gly Ser Gln Leu His Asp
Thr Phe Arg 6725 6730 6735 Phe Cys Leu Val Thr Asn Leu Thr Met Asp
Ser Val Leu Val Thr Val 6740 6745 6750 Lys Ala Leu Phe Ser Ser Asn
Leu Asp Pro Ser Leu Val Glu Gln Val 6755 6760 6765 Phe Leu Asp Lys
Thr Leu Asn Ala Ser Phe His Trp Leu Gly Ser Thr 6770 6775 6780 Tyr
Gln Leu Val Asp Ile His Val Thr Glu Met Glu Ser Ser Val Tyr 6785
6790 6795 6800 Gln Pro Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Leu
Asn Phe Thr 6805 6810 6815 Ile Thr Asn Leu Pro Tyr Ser Gln Asp Lys
Ala Gln Pro Gly Thr Thr 6820 6825 6830 Asn Tyr Gln Arg Asn Lys Arg
Asn Ile Glu Asp Ala Leu Asn Gln Leu 6835 6840 6845 Phe Arg Asn Ser
Ser Ile Lys Ser Tyr Phe Ser Asp Cys Gln Val Ser 6850 6855 6860 Thr
Phe Arg Ser Val Pro Asn Arg His His Thr Gly Val Asp Ser Leu 6865
6870 6875 6880 Cys Asn Phe Ser Pro Leu Ala Arg Arg Val Asp Arg Val
Ala Ile Tyr 6885 6890 6895 Glu Glu Phe Leu Arg Met Thr Arg Asn Gly
Thr Gln Leu Gln Asn Phe 6900 6905 6910 Thr Leu Asp Arg Ser Ser Val
Leu Val Asp Gly Tyr Ser Pro Asn Arg 6915 6920 6925 Asn Glu Pro Leu
Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala Val Ile 6930 6935 6940 Leu
Ile Gly Leu Ala Gly Leu Leu Gly Leu Ile Thr Cys Leu Ile Cys 6945
6950 6955 6960 Gly Val Leu Val Thr Thr Arg Arg Arg Lys Lys Glu Gly
Glu Tyr Asn 6965 6970 6975 Val Gln Gln Gln Cys Pro Gly Tyr Tyr Gln
Ser His Leu Asp Leu Glu 6980 6985 6990 Asp Leu Gln 6995 <210>
SEQ ID NO 5 <211> LENGTH: 622 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: MSLN <400> SEQUENCE: 5 Met Ala Leu Pro Thr
Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro 1 5 10 15 Ala Leu Gly
Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30 Pro
Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu 35 40
45 Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg
50 55 60 Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser
Thr Glu 65 70 75 80 Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys
Asn Val Lys Leu 85 90 95 Ser Thr Glu Gln Leu Arg Cys Leu Ala His
Arg Leu Ser Glu Pro Pro 100 105 110 Glu Asp Leu Asp Ala Leu Pro Leu
Asp Leu Leu Leu Phe Leu Asn Pro 115 120 125 Asp Ala Phe Ser Gly Pro
Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile 130 135 140 Thr Lys Ala Asn
Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln 145 150 155 160 Arg
Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170
175 Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu
180 185 190 Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro
Arg Leu 195 200 205 Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln
Glu Ala Ala Arg 210 215 220 Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr
Gly Pro Pro Ser Thr Trp 225 230 235 240 Ser Val Ser Thr Met Asp Ala
Leu Arg Gly Leu Leu Pro Val Leu Gly 245 250 255 Gln Pro Ile Ile Arg
Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg 260 265 270 Gln Arg Ser
Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile 275 280 285 Leu
Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295
300 Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys
305 310 315 320 Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala
Thr Gln Met 325 330 335 Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu
Gln Leu Asp Val Leu 340 345 350 Lys His Lys Leu Asp Glu Leu Tyr Pro
Gln Gly Tyr Pro Glu Ser Val 355 360 365 Ile Gln His Leu Gly Tyr Leu
Phe Leu Lys Met Ser Pro Glu Asp Ile 370 375 380 Arg Lys Trp Asn Val
Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu 385 390 395 400 Val Asn
Lys Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp 405 410 415
Arg Phe Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr 420
425 430 Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu
Glu 435 440 445 Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg
Pro Gln Asp 450 455 460 Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val
Leu Tyr Pro Lys Ala 465 470 475 480 Arg Leu Ala Phe Gln Asn Met Asn
Gly Ser Glu Tyr Phe Val Lys Ile 485 490 495 Gln Ser Phe Leu Gly Gly
Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser 500 505 510 Gln Gln Asn Val
Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr 515 520 525 Asp Ala
Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly 530 535 540
Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg 545
550 555 560 Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu
Gly Leu 565 570 575 Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val
Leu Asp Leu Ser 580 585 590 Met Gln Glu Ala Leu Ser Gly Thr Pro Cys
Leu Leu Gly Pro Gly Pro 595 600 605 Val Leu Thr Val Leu Ala Leu Leu
Leu Ala Ser Thr Leu Ala 610 615 620 <210> SEQ ID NO 6
<211> LENGTH: 690 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
SLC34A2 <400> SEQUENCE: 6 Met Ala Pro Trp Pro Glu Leu Gly Asp
Ala Gln Pro Asn Pro Asp Lys 1 5 10 15 Tyr Leu Glu Gly Ala Ala Gly
Gln Gln Pro Thr Ala Pro Asp Lys Ser 20 25 30 Lys Glu Thr Asn Lys
Thr Asp Asn Thr Glu Ala Pro Val Thr Lys Ile 35 40 45 Glu Leu Leu
Pro Ser Tyr Ser Thr Ala Thr Leu Ile Asp Glu Pro Thr 50 55 60 Glu
Val Asp Asp Pro Trp Asn Leu Pro Thr Leu Gln Asp Ser Gly Ile 65 70
75 80 Lys Trp Ser Glu Arg Asp Thr Lys Gly Lys Ile Leu Cys Phe Phe
Gln 85 90 95 Gly Ile Gly Arg Leu Ile Leu Leu Leu Gly Phe Leu Tyr
Phe Phe Val 100 105 110 Cys Ser Leu Asp Ile Leu Ser Ser Ala Phe Gln
Leu Val Gly Gly Lys 115 120 125 Met Ala Gly Gln Phe Phe Ser Asn Ser
Ser Ile Met Ser Asn Pro Leu 130 135 140 Leu Gly Leu Val Ile Gly Val
Leu Val Thr Val Leu Val Gln Ser Ser 145 150 155 160 Ser Thr Ser Thr
Ser Ile Val Val Ser Met Val Ser Ser Ser Leu Leu 165 170 175 Thr Val
Arg Ala Ala Ile Pro Ile Ile Met Gly Ala Asn Ile Gly Thr 180 185 190
Ser Ile Thr Asn Thr Ile Val Ala Leu Met Gln Val Gly Asp Arg Ser 195
200 205 Glu Phe Arg Arg Ala Phe Ala Gly Ala Thr Val His Asp Phe Phe
Asn 210 215 220 Trp Leu Ser Val Leu Val Leu Leu Pro Val Glu Val Ala
Thr His Tyr 225 230 235 240 Leu Glu Ile Ile Thr Gln Leu Ile Val Glu
Ser Phe His Phe Lys Asn 245 250 255 Gly Glu Asp Ala Pro Asp Leu Leu
Lys Val Ile Thr Lys Pro Phe Thr 260 265 270 Lys Leu Ile Val Gln Leu
Asp Lys Lys Val Ile Ser Gln Ile Ala Met 275 280 285 Asn Asp Glu Lys
Ala Lys Asn Lys Ser Leu Val Lys Ile Trp Cys Lys 290 295 300 Thr Phe
Thr Asn Lys Thr Gln Ile Asn Val Thr Val Pro Ser Thr Ala 305 310 315
320 Asn Cys Thr Ser Pro Ser Leu Cys Trp Thr Asp Gly Ile Gln Asn Trp
325 330 335 Thr Met Lys Asn Val Thr Tyr Lys Glu Asn Ile Ala Lys Cys
Gln His 340 345 350 Ile Phe Val Asn Phe His Leu Pro Asp Leu Ala Val
Gly Thr Ile Leu 355 360 365 Leu Ile Leu Ser Leu Leu Val Leu Cys Gly
Cys Leu Ile Met Ile Val 370 375 380 Lys Ile Leu Gly Ser Val Leu Lys
Gly Gln Val Ala Thr Val Ile Lys 385 390 395 400 Lys Thr Ile Asn Thr
Asp Phe Pro Phe Pro Phe Ala Trp Leu Thr Gly 405 410 415 Tyr Leu Ala
Ile Leu Val Gly Ala Gly Met Thr Phe Ile Val Gln Ser 420 425 430 Ser
Ser Val Phe Thr Ser Ala Leu Thr Pro Leu Ile Gly Ile Gly Val 435 440
445 Ile Thr Ile Glu Arg Ala Tyr Pro Leu Thr Leu Gly Ser Asn Ile Gly
450 455 460 Thr Thr Thr Thr Ala Ile Leu Ala Ala Leu Ala Ser Pro Gly
Asn Ala 465 470 475 480 Leu Arg Ser Ser Leu Gln Ile Ala Leu Cys His
Phe Phe Phe Asn Ile 485 490 495 Ser Gly Ile Leu Leu Trp Tyr Pro Ile
Pro Phe Thr Arg Leu Pro Ile 500 505 510 Arg Met Ala Lys Gly Leu Gly
Asn Ile Ser Ala Lys Tyr Arg Trp Phe 515 520 525 Ala Val Phe Tyr Leu
Ile Ile Phe Phe Phe Leu Ile Pro Leu Thr Val 530 535 540 Phe Gly Leu
Ser Leu Ala Gly Trp Arg Val Leu Val Gly Val Gly Val 545 550 555 560
Pro Val Val Phe Ile Ile Ile Leu Val Leu Cys Leu Arg Leu Leu Gln 565
570 575 Ser Arg Cys Pro Arg Val Leu Pro Lys Lys Leu Gln Asn Trp Asn
Phe 580 585 590 Leu Pro Leu Trp Met Arg Ser Leu Lys Pro Trp Asp Ala
Val Val Ser 595 600 605 Lys Phe Thr Gly Cys Phe Gln Met Arg Cys Cys
Tyr Cys Cys Arg Val 610 615 620 Cys Cys Arg Ala Cys Cys Leu Leu Cys
Gly Cys Pro Lys Cys Cys Arg 625 630 635 640 Cys Ser Lys Cys Cys Glu
Asp Leu Glu Glu Ala Gln Glu Gly Gln Asp 645 650 655 Val Pro Val Lys
Ala Pro Glu Thr Phe Asp Asn Ile Thr Ile Ser Arg 660 665 670 Glu Ala
Gln Gly Glu Val Pro Ala Ser Asp Ser Lys Thr Glu Cys Thr 675 680 685
Ala Leu 690 <210> SEQ ID NO 7 <211> LENGTH: 1093
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: KIAA1445
<400> SEQUENCE: 7 Met Val Leu Ala Gly Pro Leu Ala Val Ser Leu
Leu Leu Pro Ser Leu 1 5 10 15 Thr Leu Leu Val Ser His Leu Ser Ser
Ser Gln Asp Val Ser Ser Glu 20 25 30 Pro Ser Ser Glu Gln Gln Leu
Cys Ala Leu Ser Lys His Pro Thr Val 35 40 45 Ala Phe Glu Asp Leu
Gln Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly 50 55 60 Ala Arg Asp
Phe Ser Gln Leu Ala Leu Asp Pro Ser Gly Asn Gln Leu 65 70 75 80 Ile
Val Gly Ala Arg Asn Tyr Leu Phe Arg Leu Ser Leu Ala Asn Val 85 90
95 Ser Leu Leu Gln Ala Thr Glu Trp Ala Ser Ser Glu Asp Thr Arg Arg
100 105 110 Ser Cys Gln Ser Lys Gly Lys Thr Glu Glu Glu Cys Gln Asn
Tyr Val 115 120 125 Arg Val Leu Ile Val Ala Gly Arg Lys Val Phe Met
Cys Gly Thr Asn 130 135 140 Ala Phe Ser Pro Met Cys Thr Ser Arg Gln
Val Gly Asn Leu Ser Arg 145 150 155 160 Thr Thr Glu Lys Ile Asn Gly
Val Ala Arg Cys Pro Tyr Asp Pro Arg 165 170 175 His Asn Ser Thr Ala
Val Ile Ser Ser Gln Gly Glu Leu Tyr Ala Ala 180 185 190 Thr Val Ile
Asp Phe Ser Gly Arg Asp Pro Ala Ile Tyr Arg Ser Leu 195 200 205 Gly
Ser Gly Pro Pro Leu Arg Thr Ala Gln Tyr Asn Ser Lys Trp Leu 210 215
220 Asn Glu Pro Asn Phe Val Ala Ala Tyr Asp Ile Gly Leu Phe Ala Tyr
225 230 235 240 Phe Phe Leu Arg Glu Asn Ala Val Glu His Asp Cys Gly
Arg Thr Val 245 250 255 Tyr Ser Arg Val Ala Arg Val Cys Lys Asn Asp
Val Gly Gly Arg Phe 260 265 270 Leu Leu Glu Asp Thr Trp Thr Thr Phe
Met Lys Ala Arg Leu Asn Cys 275 280 285 Ser Arg Pro Gly Glu Val Pro
Phe Tyr Tyr Asn Glu Leu Gln Ser Ala 290 295 300 Phe His Leu Pro Glu
Gln Asp Leu Ile Tyr Gly Val Phe Thr Thr Asn 305 310 315 320 Val Asn
Ser Ile Ala Ala Ser Ala Val Cys Ala Phe Asn Leu Ser Ala 325 330 335
Ile Ser Gln Ala Phe Asn Gly Pro Phe Arg Tyr Gln Glu Asn Pro Arg 340
345 350 Ala Ala Trp Leu Pro Ile Ala Asn Pro Ile Pro Asn Phe Gln Cys
Gly 355 360 365 Thr Leu Pro Glu Thr Gly Pro Asn Glu Asn Leu Thr Glu
Arg Ser Leu 370 375 380 Gln Asp Ala Gln Arg Leu Phe Leu Met Ser Glu
Ala Val Gln Pro Val 385 390 395 400 Thr Pro Glu Pro Cys Val Thr Gln
Asp Ser Val Arg Phe Ser His Leu 405 410 415 Val Val Asp Leu Val Gln
Ala Lys Asp Thr Leu Tyr His Val Leu Tyr 420 425 430 Ile Gly Thr Glu
Ser Gly Thr Ile Leu Lys Ala Leu Ser Thr Ala Ser 435 440 445 Arg Ser
Leu His Gly Cys Tyr Leu Glu Glu Leu His Val Leu Pro Pro 450 455 460
Gly Arg Arg Glu Pro Leu Arg Ser Leu Arg Ile Leu His Ser Ala Arg 465
470 475 480 Ala Leu Phe Val Gly Leu Arg Asp Gly Val Leu Arg Val Pro
Leu Glu 485 490 495 Arg Cys Ala Ala Tyr Arg Ser Gln Gly Ala Cys Leu
Gly Ala Arg Asp 500 505 510 Pro Tyr Cys Gly Trp Asp Gly Lys Gln Gln
Arg Cys Ser Thr Leu Glu 515 520 525 Asp Ser Ser Asn Met Ser Leu Trp
Thr Gln Asn Ile Thr Ala Cys Pro 530 535 540 Val Arg Asn Val Thr Arg
Asp Gly Gly Phe Gly Pro Trp Ser Pro Trp 545 550 555 560 Gln Pro Cys
Glu His Leu Asp Gly Asp Asn Ser Gly Ser Cys Leu Cys 565 570 575 Arg
Ala Arg Ser Cys Asp Ser Pro Arg Pro Arg Cys Gly Gly Leu Asp 580 585
590 Cys Leu Gly Pro Ala Ile His Ile Ala Asn Cys Ser Arg Asn Gly Ala
595 600 605 Trp Thr Pro Trp Ser Ser Trp Ala Leu Cys Ser Thr Ser Cys
Gly Ile 610 615 620 Gly Phe Gln Val Arg Gln Arg Ser Cys Ser Asn Pro
Ala Pro Arg His 625 630 635 640 Gly Gly Arg Ile Cys Val Gly Lys Ser
Arg Glu Glu Arg Phe Cys Asn 645 650 655 Glu Asn Thr Pro Cys Pro Val
Pro Ile Phe Trp Ala Ser Trp Gly Ser 660 665 670 Trp Ser Lys Cys Ser
Ser Asn Cys Gly Gly Gly Met Gln Ser Arg Arg 675 680 685 Arg Ala Cys
Glu Asn Gly Asn Ser Cys Leu Gly Cys Gly Val Glu Phe 690 695 700 Lys
Thr Cys Asn Pro Glu Gly Cys Pro Glu Val Arg Arg Asn Thr Pro 705 710
715 720 Trp Thr Pro Trp Leu Pro Val Asn Val Thr Gln Gly Gly Ala Arg
Gln 725 730 735 Glu Gln Arg Phe Arg Phe Thr Cys Arg Ala Pro Leu Ala
Asp Pro His 740 745 750 Gly Leu Gln Phe Gly Arg Arg Arg Thr Glu Thr
Arg Thr Cys Pro Ala 755 760 765 Asp Gly Ser Gly Ser Cys Asp Thr Asp
Ala Leu Val Glu Asp Leu Leu 770 775 780 Arg Ser Gly Ser Thr Ser Pro
His Thr Val Ser Gly Gly Trp Ala Ala 785 790 795 800 Trp Gly Pro Trp
Ser Ser Cys Ser Arg Asp Cys Glu Leu Gly Phe Arg 805 810 815 Val Arg
Lys Arg Thr Cys Thr Asn Pro Glu Pro Arg Asn Gly Gly Leu 820 825 830
Pro Cys Val Gly Asp Ala Ala Glu Tyr Gln Asp Cys Asn Pro Gln Ala 835
840 845 Cys Pro Val Arg Gly Ala Trp Ser Cys Trp Thr Ser Trp Ser Pro
Cys 850 855 860 Ser Ala Ser Cys Gly Gly Gly His Tyr Gln Arg Thr Arg
Ser Cys Thr 865 870 875 880 Ser Pro Ala Pro Ser Pro Gly Glu Asp Ile
Cys Leu Gly Leu His Thr 885 890 895 Glu Glu Ala Leu Cys Ala Thr Gln
Ala Cys Pro Glu Gly Trp Ser Pro 900 905 910 Trp Ser Glu Trp Ser Lys
Cys Thr Asp Asp Gly Ala Gln Ser Arg Ser 915 920 925 Arg His Cys Glu
Glu Leu Leu Pro Gly Ser Ser Ala Cys Ala Gly Asn 930 935 940 Ser Ser
Gln Ser Arg Pro Cys Pro Tyr Ser Glu Ile Pro Val Ile Leu 945 950 955
960 Pro Ala Ser Ser Met Glu Glu Ala Thr Gly Cys Ala Gly Phe Asn Leu
965 970 975 Ile His Leu Val Ala Thr Gly Ile Ser Cys Phe Leu Gly Ser
Gly Leu 980 985 990 Leu Thr Leu Ala Val Tyr Leu Ser Cys Gln His Cys
Gln Arg Gln Ser 995 1000 1005 Gln Glu Ser Thr Leu Val His Pro Ala
Thr Pro Asn His Leu His Tyr 1010 1015 1020 Lys Gly Gly Gly Thr Pro
Lys Asn Glu Lys Tyr Thr Pro Met Glu Phe 1025 1030 1035 1040 Lys Thr
Leu Asn Lys Asn Asn Leu Ile Pro Asp Asp Arg Ala Asn Phe 1045 1050
1055 Tyr Pro Leu Gln Gln Thr Asn Val Tyr Thr Thr Thr Tyr Tyr Pro
Ser 1060 1065 1070 Pro Leu Asn Lys His Ser Phe Arg Pro Glu Ala Ser
Pro Gly Gln Arg 1075 1080 1085 Cys Phe Pro Asn Ser 1090 <210>
SEQ ID NO 8 <211> LENGTH: 141 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: PSCA hlg <400> SEQUENCE: 8 Met Trp Val Leu
Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu Leu 1 5 10 15 Pro Gly
Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu Phe Gln 20 25 30
Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn Cys Thr Val 35
40 45 Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu Gln Ser Ala
Gly 50 55 60 Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala Ala Cys
Leu Ile Ala 65 70 75 80 Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro Gly
Lys Leu Asn Ser Val 85 90 95 Cys Ile Ser Cys Cys Asn Thr Pro Leu
Cys Asn Gly Pro Arg Pro Lys 100 105 110 Lys Arg Gly Ser Ser Ala Ser
Ala Leu Arg Pro Gly Leu Arg Thr Thr 115 120 125 Ile Leu Phe Leu Lys
Leu Ala Leu Phe Ser Ala His Cys 130 135 140 <210> SEQ ID NO 9
<211> LENGTH: 442 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
ETBR <400> SEQUENCE: 9 Met Gln Pro Pro Pro Ser Leu Cys Gly
Arg Ala Leu Val Ala Leu Val 1 5 10 15 Leu Ala Cys Gly Leu Ser Arg
Ile Trp Gly Glu Glu Arg Gly Phe Pro 20 25 30 Pro Asp Arg Ala Thr
Pro Leu Leu Gln Thr Ala Glu Ile Met Thr Pro 35 40 45 Pro Thr Lys
Thr Leu Trp Pro Lys Gly Ser Asn Ala Ser Leu Ala Arg 50 55 60 Ser
Leu Ala Pro Ala Glu Val Pro Lys Gly Asp Arg Thr Ala Gly Ser 65 70
75 80 Pro Pro Arg Thr Ile Ser Pro Pro Pro Cys Gln Gly Pro Ile Glu
Ile 85 90 95 Lys Glu Thr Phe Lys Tyr Ile Asn Thr Val Val Ser Cys
Leu Val Phe 100 105 110 Val Leu Gly Ile Ile Gly Asn Ser Thr Leu Leu
Arg Ile Ile Tyr Lys 115 120 125 Asn Lys Cys Met Arg Asn Gly Pro Asn
Ile Leu Ile Ala Ser Leu Ala 130 135 140 Leu Gly Asp Leu Leu His Ile
Val Ile Asp Ile Pro Ile Asn Val Tyr 145 150 155 160 Lys Leu Leu Ala
Glu Asp Trp Pro Phe Gly Ala Glu Met Cys Lys Leu 165 170 175 Val Pro
Phe Ile Gln Lys Ala Ser Val Gly Ile Thr Val Leu Ser Leu 180 185 190
Cys Ala Leu Ser Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg 195
200 205 Ile Lys Gly Ile Gly Val Pro Lys Trp Thr Ala Val Glu Ile Val
Leu 210 215 220 Ile Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala
Ile Gly Phe 225 230 235 240 Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser
Tyr Leu Arg Ile Cys Leu 245 250 255 Leu His Pro Val Gln Lys Thr Ala
Phe Met Gln Phe Tyr Lys Thr Ala 260 265 270 Lys Asp Trp Trp Leu Phe
Ser Phe Tyr Phe Cys Leu Pro Leu Ala Ile 275 280 285 Thr Ala Phe Phe
Tyr Thr Leu Met Thr Cys Glu Met Leu Arg Lys Lys 290 295 300 Ser Gly
Met Gln Ile Ala Leu Asn Asp His Leu Lys Gln Arg Arg Glu 305 310 315
320 Val Ala Lys Thr Val Phe Cys Leu Val Leu Val Phe Ala Leu Cys Trp
325 330 335 Leu Pro Leu His Leu Ser Arg Ile Leu Lys Leu Thr Leu Tyr
Asn Gln 340 345 350 Asn Asp Pro Asn Arg Cys Glu Leu Leu Ser Phe Leu
Leu Val Leu Asp 355 360 365 Tyr Ile Gly Ile Asn Met Ala Ser Leu Asn
Ser Cys Ile Asn Pro Ile 370 375 380 Ala Leu Tyr Leu Val Ser Lys Arg
Phe Lys Asn Cys Phe Lys Ser Cys 385 390 395 400 Leu Cys Cys Trp Cys
Gln Ser Phe Glu Glu Lys Gln Ser Leu Glu Glu 405 410 415 Lys Gln Ser
Cys Leu Lys Phe Lys Ala Asn Asp His Gly Tyr Asp Asn 420 425 430 Phe
Arg Ser Ser Asn Lys Tyr Ser Ser Ser 435 440 <210> SEQ ID NO
10 <211> LENGTH: 783 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: RNF124 <400> SEQUENCE: 10 Met Ser Gly Gly His
Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu 1 5 10 15 Leu Met Ala
Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu Val Leu 20 25 30 Ala
Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile 35 40
45 Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn Leu Thr
50 55 60 Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala
Glu Gly 65 70 75 80 Lys Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn
Ala Ser Asp Asp 85 90 95 Asp Asn Leu Glu Pro Gly Phe Ile Ser Ile
Val Lys Leu Glu Ser Pro 100 105 110 Arg Arg Ala Pro Arg Pro Cys Leu
Ser Leu Ala Ser Lys Ala Arg Met 115 120 125 Ala Gly Glu Arg Gly Ala
Ser Ala Val Leu Phe Asp Ile Thr Glu Asp 130 135 140 Arg Ala Ala Ala
Glu Gln Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro 145 150 155 160 Val
Val Leu Ile Trp Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val 165 170
175 Tyr Lys Asn Gln Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro
180 185 190 Ala Trp Pro Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val
Gly Thr 195 200 205 Ile Phe Val Ile Ile Leu Ala Ser Val Leu Arg Ile
Arg Cys Arg Pro 210 215 220 Arg His Ser Arg Pro Asp Pro Leu Gln Gln
Arg Thr Ala Trp Ala Ile 225 230 235 240 Ser Gln Leu Ala Thr Arg Arg
Tyr Gln Ala Ser Cys Arg Gln Ala Arg 245 250 255 Gly Glu Trp Pro Asp
Ser Gly Ser Ser Cys Ser Ser Ala Pro Val Cys 260 265 270 Ala Ile Cys
Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu Arg Val Ile 275 280 285 Ser
Cys Leu His Glu Phe His Arg Asn Cys Val Asp Pro Trp Leu His 290 295
300 Gln His Arg Thr Cys Pro Leu Cys Val Phe Asn Ile Thr Glu Gly Asp
305 310 315 320 Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg Ser Tyr Gln
Glu Pro Gly 325 330 335 Arg Arg Leu His Leu Ile Arg Gln His Pro Gly
His Ala His Tyr His 340 345 350 Leu Pro Ala Ala Tyr Leu Leu Gly Pro
Ser Arg Ser Ala Val Ala Arg 355 360 365 Pro Pro Arg Pro Gly Pro Phe
Leu Pro Ser Gln Glu Pro Gly Met Gly 370 375 380 Pro Arg His His Arg
Phe Pro Arg Ala Ala His Pro Arg Ala Pro Gly 385 390 395 400 Glu Gln
Gln Arg Leu Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp 405 410 415
Gly Met Ser His Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro 420
425 430 Val Pro Leu Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly
Glu 435 440 445 Ser Tyr Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly
Pro Ala Ser 450 455 460 Asp Ser Ser Ser Gly Pro Cys His Gly Ser Ser
Ser Asp Ser Val Val 465 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln
Gly Val His Gly Ser Ser Ser Thr 485 490 495 Phe Cys Ser Ser Leu Ser
Ser Asp Phe Asp Pro Leu Val Tyr Cys Ser 500 505 510 Pro Lys Gly Asp
Pro Gln Arg Val Asp Met Gln Pro Ser Val Thr Ser 515 520 525 Arg Pro
Arg Ser Leu Asp Ser Val Val Pro Thr Gly Glu Thr Gln Val 530 535 540
Ser Ser His Val His Tyr His Arg His Arg His His His Tyr Lys Lys 545
550 555 560 Arg Phe Gln Trp His Gly Arg Lys Pro Gly Pro Glu Thr Gly
Val Pro 565 570 575 Gln Ser Arg Pro Pro Ile Pro Arg Thr Gln Pro Gln
Pro Glu Pro Pro 580 585 590 Ser Pro Asp Gln Gln Val Thr Gly Ser Asn
Ser Ala Ala Pro Ser Gly 595 600 605 Arg Leu Ser Asn Pro Gln Cys Pro
Arg Ala Leu Pro Glu Pro Ala Pro 610 615 620 Gly Pro Val Asp Ala Ser
Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe 625 630 635 640 Asn Leu Gln
Lys Ser Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg 645 650 655 Arg
Gly Gly Pro Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala 660 665
670 Thr Val His Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val
675 680 685 Ala Tyr Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly
Pro Pro 690 695 700 Gly Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly
Pro Cys Tyr Ser 705 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu
Thr Pro Arg Gln Pro Leu Glu 725 730 735 Pro His Pro Pro Gly Glu Gly
Pro Ser Glu Trp Ser Ser Asp Thr Ala 740 745 750 Glu Gly Arg Pro Cys
Pro Tyr Pro His Cys Gln Val Leu Ser Ala Gln 755 760 765 Pro Gly Ser
Glu Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala Val 770 775 780
<210> SEQ ID NO 11 <211> LENGTH: 490 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: STEAP2 <400> SEQUENCE: 11 Met
Glu Ser Ile Ser Met Met Gly Ser Pro Lys Ser Leu Ser Glu Thr 1 5 10
15 Val Leu Pro Asn Gly Ile Asn Gly Ile Lys Asp Ala Arg Lys Val Thr
20 25 30 Val Gly Val Ile Gly Ser Gly Asp Phe Ala Lys Ser Leu Thr
Ile Arg 35 40 45 Leu Ile Arg Cys Gly Tyr His Val Val Ile Gly Ser
Arg Asn Pro Lys 50 55 60 Phe Ala Ser Glu Phe Phe Pro His Val Val
Asp Val Thr His His Glu 65 70 75 80 Asp Ala Leu Thr Lys Thr Asn Ile
Ile Phe Val Ala Ile His Arg Glu 85 90 95 His Tyr Thr Ser Leu Trp
Asp Leu Arg His Leu Leu Val Gly Lys Ile 100 105 110 Leu Ile Asp Val
Ser Asn Asn Met Arg Ile Asn Gln Tyr Pro Glu Ser 115 120 125 Asn Ala
Glu Tyr Leu Ala Ser Leu Phe Pro Asp Ser Leu Ile Val Lys 130 135 140
Gly Phe Asn Val Val Ser Ala Trp Ala Leu Gln Leu Gly Pro Lys Asp 145
150 155 160 Ala Ser Arg Gln Val Tyr Ile Cys Ser Asn Asn Ile Gln Ala
Arg Gln 165 170 175 Gln Val Ile Glu Leu Ala Arg Gln Leu Asn Phe Ile
Pro Ile Asp Leu 180 185 190 Gly Ser Leu Ser Ser Ala Arg Glu Ile Glu
Asn Leu Pro Leu Arg Leu 195 200 205 Phe Thr Leu Trp Arg Gly Pro Val
Val Val Ala Ile Ser Leu Ala Thr 210 215 220 Phe Phe Phe Leu Tyr Ser
Phe Val Arg Asp Val Ile His Pro Tyr Ala 225 230 235 240 Arg Asn Gln
Gln Ser Asp Phe Tyr Lys Ile Pro Ile Glu Ile Val Asn 245 250 255 Lys
Thr Leu Pro Ile Val Ala Ile Thr Leu Leu Ser Leu Val Tyr Leu 260 265
270 Ala Gly Leu Leu Ala Ala Ala Tyr Gln Leu Tyr Tyr Gly Thr Lys Tyr
275 280 285 Arg Arg Phe Pro Pro Trp Leu Glu Thr Trp Leu Gln Cys Arg
Lys Gln 290 295 300 Leu Gly Leu Leu Ser Phe Phe Phe Ala Met Val His
Val Ala Tyr Ser 305 310 315 320 Leu Cys Leu Pro Met Arg Arg Ser Glu
Arg Tyr Leu Phe Leu Asn Met 325 330 335 Ala Tyr Gln Gln Val His Ala
Asn Ile Glu Asn Ser Trp Asn Glu Glu 340 345 350 Glu Val Trp Arg Ile
Glu Met Tyr Ile Ser Phe Gly Ile Met Ser Leu 355 360 365 Gly Leu Leu
Ser Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asn 370 375 380 Ala
Leu Asn Trp Arg Glu Phe Ser Phe Ile Gln Ser Thr Leu Gly Tyr 385 390
395 400 Val Ala Leu Leu Ile Ser Thr Phe His Val Leu Ile Tyr Gly Trp
Lys 405 410 415 Arg Ala Phe Glu Glu Glu Tyr Tyr Arg Phe Tyr Thr Pro
Pro Asn Phe 420 425 430 Val Leu Ala Leu Val Leu Pro Ser Ile Val Ile
Leu Gly Lys Ile Ile 435 440 445 Leu Phe Leu Pro Cys Ile Ser Gln Lys
Leu Lys Arg Ile Lys Lys Gly 450 455 460 Trp Glu Lys Ser Gln Phe Leu
Glu Glu Gly Ile Gly Gly Thr Ile Pro 465 470 475 480 His Val Ser Pro
Glu Arg Val Thr Val Met 485 490 <210> SEQ ID NO 12
<211> LENGTH: 1214 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: TrpM4 <400> SEQUENCE: 12 Met Val Val Pro Glu Lys
Glu Gln Ser Trp Ile Pro Lys Ile Phe Lys 1 5 10 15 Lys Lys Thr Cys
Thr Thr Phe Ile Val Asp Ser Thr Asp Pro Gly Gly 20 25 30 Thr Leu
Cys Gln Cys Gly Arg Pro Arg Thr Ala His Pro Ala Val Ala 35 40 45
Met Glu Asp Ala Phe Gly Ala Ala Val Val Thr Val Trp Asp Ser Asp 50
55 60 Ala His Thr Thr Glu Lys Pro Thr Asp Ala Tyr Gly Glu Leu Asp
Phe 65 70 75 80 Thr Gly Ala Gly Arg Lys His Ser Asn Phe Leu Arg Leu
Ser Asp Arg 85 90 95 Thr Asp Pro Ala Ala Val Tyr Ser Leu Val Thr
Arg Thr Trp Gly Phe 100 105 110 Arg Ala Pro Asn Leu Val Val Ser Val
Leu Gly Gly Ser Gly Gly Pro 115 120 125 Val Leu Gln Thr Trp Leu Gln
Asp Leu Leu Arg Arg Gly Leu Val Arg 130 135 140 Ala Ala Gln Ser Thr
Gly Ala Trp Ile Val Thr Gly Gly Leu His Thr 145 150 155 160 Gly Ile
Gly Arg His Val Gly Val Ala Val Arg Asp His Gln Met Ala 165 170 175
Ser Thr Gly Gly Thr Lys Val Val Ala Met Gly Val Ala Pro Trp Gly 180
185 190 Val Val Arg Asn Arg Asp Thr Leu Ile Asn Pro Lys Gly Ser Phe
Pro 195 200 205 Ala Arg Tyr Arg Trp Arg Gly Asp Pro Glu Asp Gly Val
Gln Phe Pro 210 215 220 Leu Asp Tyr Asn Tyr Ser Ala Phe Phe Leu Val
Asp Asp Gly Thr His 225 230 235 240 Gly Cys Leu Gly Gly Glu Asn Arg
Phe Arg Leu Arg Leu Glu Ser Tyr 245 250 255 Ile Ser Gln Gln Lys Thr
Gly Val Gly Gly Thr Gly Ile Asp Ile Pro 260 265 270 Val Leu Leu Leu
Leu Ile Asp Gly Asp Glu Lys Met Leu Thr Arg Ile 275 280 285 Glu Asn
Ala Thr Gln Ala Gln Leu Pro Cys Leu Leu Val Ala Gly Ser 290 295 300
Gly Gly Ala Ala Asp Cys Leu Ala Glu Thr Leu Glu Asp Thr Leu Ala 305
310 315 320 Pro Gly Ser Gly Gly Ala Arg Gln Gly Glu Ala Arg Asp Arg
Ile Arg 325 330 335 Arg Phe Phe Pro Lys Gly Asp Leu Glu Val Leu Gln
Ala Gln Val Glu 340 345 350 Arg Ile Met Thr Arg Lys Glu Leu Leu Thr
Val Tyr Ser Ser Glu Asp 355 360 365 Gly Ser Glu Glu Phe Glu Thr Ile
Val Leu Lys Ala Leu Val Lys Ala 370 375 380 Cys Gly Ser Ser Glu Ala
Ser Ala Tyr Leu Asp Glu Leu Arg Leu Ala 385 390 395 400 Val Ala Trp
Asn Arg Val Asp Ile Ala Gln Ser Glu Leu Phe Arg Gly 405 410 415 Asp
Ile Gln Trp Arg Ser Phe His Leu Glu Ala Ser Leu Met Asp Ala 420 425
430 Leu Leu Asn Asp Arg Pro Glu Phe Val Arg Leu Leu Ile Ser His Gly
435 440 445 Leu Ser Leu Gly His Phe Leu Thr Pro Met Arg Leu Ala Gln
Leu Tyr 450 455 460 Ser Ala Ala Pro Ser Asn Ser Leu Ile Arg Asn Leu
Leu Asp Gln Ala 465 470 475 480 Ser His Ser Ala Gly Thr Lys Ala Pro
Ala Leu Lys Gly Gly Ala Ala 485 490 495 Glu Leu Arg Pro Pro Asp Val
Gly His Val Leu Arg Met Leu Leu Gly 500 505 510 Lys Met Cys Ala Pro
Arg Tyr Pro Ser Gly Gly Ala Trp Asp Pro His 515 520 525 Pro Gly Gln
Gly Phe Gly Glu Ser Met Tyr Leu Leu Ser Asp Lys Ala 530 535 540 Thr
Ser Pro Leu Ser Leu Asp Ala Gly Leu Gly Gln Ala Pro Trp Ser 545 550
555 560 Asp Leu Leu Leu Trp Ala Leu Leu Leu Asn Arg Ala Gln Met Ala
Met 565 570 575 Tyr Phe Trp Glu Met Gly Ser Asn Ala Val Ser Ser Ala
Leu Gly Ala 580 585 590 Cys Leu Leu Leu Arg Val Met Ala Arg Leu Glu
Pro Asp Ala Glu Glu 595 600 605 Ala Ala Arg Arg Lys Asp Leu Ala Phe
Lys Phe Glu Gly Met Gly Val 610 615 620 Asp Leu Phe Gly Glu Cys Tyr
Arg Ser Ser Glu Val Arg Ala Ala Arg 625 630 635 640 Leu Leu Leu Arg
Arg Cys Pro Leu Trp Gly Asp Ala Thr Cys Leu Gln 645 650 655 Leu Ala
Met Gln Ala Asp Ala Arg Ala Phe Phe Ala Gln Asp Gly Val 660 665 670
Gln Ser Leu Leu Thr Gln Lys Trp Trp Gly Asp Met Ala Ser Thr Thr 675
680 685 Pro Ile Trp Ala Leu Val Leu Ala Phe Phe Cys Pro Pro Leu Ile
Tyr 690 695 700 Thr Arg Leu Ile Thr Phe Arg Lys Ser Glu Glu Glu Pro
Thr Arg Glu 705 710 715 720 Glu Leu Glu Phe Asp Met Asp Ser Val Ile
Asn Gly Glu Gly Pro Val 725 730 735 Gly Thr Ala Asp Pro Ala Glu Lys
Thr Pro Leu Gly Val Pro Arg Gln 740 745 750 Ser Gly Arg Pro Gly Cys
Cys Gly Gly Arg Cys Gly Gly Arg Arg Cys 755 760 765 Leu Arg Arg Trp
Phe His Phe Trp Gly Ala Pro Val Thr Ile Phe Met 770 775 780 Gly Asn
Val Val Ser Tyr Leu Leu Phe Leu Leu Leu Phe Ser Arg Val 785 790 795
800 Leu Leu Val Asp Phe Gln Pro Ala Pro Pro Gly Ser Leu Glu Leu Leu
805 810 815 Leu Tyr Phe Trp Ala Phe Thr Leu Leu Cys Glu Glu Leu Arg
Gln Gly 820 825 830 Leu Ser Gly Gly Gly Gly Ser Leu Ala Ser Gly Gly
Pro Gly Pro Gly 835 840 845 His Ala Ser Leu Ser Gln Arg Leu Arg Leu
Tyr Leu Ala Asp Ser Trp 850 855 860 Asn Gln Cys Asp Leu Val Ala Leu
Thr Cys Phe Leu Leu Gly Val Gly 865 870 875 880 Cys Arg Leu Thr Pro
Gly Leu Tyr His Leu Gly Arg Thr Val Leu Cys 885 890 895 Ile Asp Phe
Met Val Phe Thr Val Arg Leu Leu His Ile Phe Thr Val 900 905 910 Asn
Lys Gln Leu Gly Pro Lys Ile Val Ile Val Ser Lys Met Met Lys 915 920
925 Asp Val Phe Phe Phe Leu Phe Phe Leu Gly Val Trp Leu Val Ala Tyr
930 935 940 Gly Val Ala Thr Glu Gly Leu Leu Arg Pro Arg Asp Ser Asp
Phe Pro 945 950 955 960 Ser Ile Leu Arg Arg Val Phe Tyr Arg Pro Tyr
Leu Gln Ile Phe Gly 965 970 975 Gln Ile Pro Gln Glu Asp Met Asp Val
Ala Leu Met Glu His Ser Asn 980 985 990 Cys Ser Ser Glu Pro Gly Phe
Trp Ala His Pro Pro Gly Ala Gln Ala 995 1000 1005 Gly Thr Cys Val
Ser Gln Tyr Ala Asn Trp Leu Val Val Leu Leu Leu 1010 1015 1020 Val
Ile Phe Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile 1025
1030 1035 1040 Ala Met Phe Ser Tyr Thr Phe Gly Lys Val Gln Gly Asn
Ser Asp Leu 1045 1050 1055 Tyr Trp Lys Ala Gln Arg Tyr Arg Leu Ile
Arg Glu Phe His Ser Arg 1060 1065 1070 Pro Ala Leu Ala Pro Pro Phe
Ile Val Ile Ser His Leu Arg Leu Leu 1075 1080 1085 Leu Arg Gln Leu
Cys Arg Arg Pro Arg Ser Pro Gln Pro Ser Ser Pro 1090 1095 1100 Ala
Leu Glu His Phe Arg Val Tyr Leu Ser Lys Glu Ala Glu Arg Lys 1105
1110 1115 1120 Leu Leu Thr Trp Glu Ser Val His Lys Glu Asn Phe Leu
Leu Ala Arg 1125 1130 1135 Ala Arg Asp Lys Arg Glu Ser Asp Ser Glu
Arg Leu Lys Arg Thr Ser 1140 1145 1150 Gln Lys Val Asp Leu Ala Leu
Lys Gln Leu Gly His Ile Arg Glu Tyr 1155 1160 1165 Glu Gln Arg Leu
Lys Val Leu Glu Arg Glu Val Gln Gln Cys Ser Arg 1170 1175 1180 Val
Leu Gly Trp Val Ala Glu Ala Leu Ser Arg Ser Ala Leu Leu Pro 1185
1190 1195 1200 Pro Gly Gly Pro Pro Pro Pro Asp Leu Pro Gly Ser Lys
Asp 1205 1210 <210> SEQ ID NO 13 <211> LENGTH: 188
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: TDGF1
<400> SEQUENCE: 13 Met Asp Cys Arg Lys Met Ala Arg Phe Ser
Tyr Ser Val Ile Trp Ile 1 5 10 15 Met Ala Ile Ser Lys Val Phe Glu
Leu Gly Leu Val Ala Gly Leu Gly 20 25 30 His Gln Glu Phe Ala Arg
Pro Ser Arg Gly Tyr Leu Ala Phe Arg Asp 35 40 45 Asp Ser Ile Trp
Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg Ser Ser 50 55 60 Gln Arg
Val Pro Pro Met Gly Ile Gln His Ser Lys Glu Leu Asn Arg 65 70 75 80
Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly Ser Phe Cys Ala 85
90 95 Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu His Asp Val Arg
Lys 100 105 110 Glu Asn Cys Gly Ser Val Pro His Asp Thr Trp Leu Pro
Lys Lys Cys 115 120 125 Ser Leu Cys Lys Cys Trp His Gly Gln Leu Arg
Cys Phe Pro Gln Ala 130 135 140 Phe Leu Pro Gly Cys Asp Gly Leu Val
Met Asp Glu His Leu Val Ala 145 150 155 160 Ser Arg Thr Pro Glu Leu
Pro Pro Ser Ala Arg Thr Thr Thr Phe Met 165 170 175 Leu Val Gly Ile
Cys Leu Ser Ile Gln Ser Tyr Tyr 180 185 <210> SEQ ID NO 14
<211> LENGTH: 1033 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: CD21 <400> SEQUENCE: 14 Met Gly Ala Ala Gly Leu
Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly
Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile
Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45
Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50
55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala
Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu
Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro
Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn
Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn
Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val Ser Val
Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile 145 150 155 160 His Asn
Gly His His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly 165 170 175
Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu 180
185 190 Lys Ile Ile Asn Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro
Pro 195 200 205 Thr Cys Glu Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe
Pro Asn Gly 210 215 220 Lys Val Lys Glu Pro Pro Ile Leu Arg Val Gly
Val Thr Ala Asn Phe 225 230 235 240 Phe Cys Asp Glu Gly Tyr Arg Leu
Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255 Val Ile Ala Gly Gln Gly
Val Ala Trp Thr Lys Met Pro Val Cys Glu 260 265 270 Glu Ile Phe Cys
Pro Ser Pro Pro Pro Ile Leu Asn Gly Arg His Ile 275 280 285 Gly Asn
Ser Leu Ala Asn Val Ser Tyr Gly Ser Ile Val Thr Tyr Thr 290 295 300
Cys Asp Pro Asp Pro Glu Glu Gly Val Asn Phe Ile Leu Ile Gly Glu 305
310 315 320 Ser Thr Leu Arg Cys Thr Val Asp Ser Gln Lys Thr Gly Thr
Trp Ser 325 330 335 Gly Pro Ala Pro Arg Cys Glu Leu Ser Thr Ser Ala
Val Gln Cys Pro 340 345 350 His Pro Gln Ile Leu Arg Gly Arg Met Val
Ser Gly Gln Lys Asp Arg 355 360 365 Tyr Thr Tyr Asn Asp Thr Val Ile
Phe Ala Cys Met Phe Gly Phe Thr 370 375 380 Leu Lys Gly Ser Lys Gln
Ile Arg Cys Asn Ala Gln Gly Thr Trp Glu 385 390 395 400 Pro Ser Ala
Pro Val Cys Glu Lys Glu Cys Gln Ala Pro Pro Asn Ile 405 410 415 Leu
Asn Gly Gln Lys Glu Asp Arg His Met Val Arg Phe Asp Pro Gly 420 425
430 Thr Ser Ile Lys Tyr Ser Cys Asn Pro Gly Tyr Val Leu Val Gly Glu
435 440 445 Glu Ser Ile Gln Cys Thr Ser Glu Gly Val Trp Thr Pro Pro
Val Pro 450 455 460 Gln Cys Lys Val Ala Ala Cys Glu Ala Thr Gly Arg
Gln Leu Leu Thr 465 470 475 480 Lys Pro Gln His Gln Phe Val Arg Pro
Asp Val Asn Ser Ser Cys Gly 485 490 495 Glu Gly Tyr Lys Leu Ser Gly
Ser Val Tyr Gln Glu Cys Gln Gly Thr 500 505 510 Ile Pro Trp Phe Met
Glu Ile Arg Leu Cys Lys Glu Ile Thr Cys Pro 515 520 525 Pro Pro Pro
Val Ile Tyr Asn Gly Ala His Thr Gly Ser Ser Leu Glu 530 535 540 Asp
Phe Pro Tyr Gly Thr Thr Val Thr Tyr Thr Cys Asn Pro Gly Pro 545 550
555 560 Glu Arg Gly Val Glu Phe Ser Leu Ile Gly Glu Ser Thr Ile Arg
Cys 565 570 575 Thr Ser Asn Asp Gln Glu Arg Gly Thr Trp Ser Gly Pro
Ala Pro Leu 580 585 590 Cys Lys Leu Ser Leu Leu Ala Val Gln Cys Ser
His Val His Ile Ala 595 600 605 Asn Gly Tyr Lys Ile Ser Gly Lys Glu
Ala Pro Tyr Phe Tyr Asn Asp 610 615 620 Thr Val Thr Phe Lys Cys Tyr
Ser Gly Phe Thr Leu Lys Gly Ser Ser 625 630 635 640 Gln Ile Arg Cys
Lys Ala Asp Asn Thr Trp Asp Pro Glu Ile Pro Val 645 650 655 Cys Glu
Lys Glu Thr Cys Gln His Val Arg Gln Ser Leu Gln Glu Leu 660 665 670
Pro Ala Gly Ser Arg Val Glu Leu Val Asn Thr Ser Cys Gln Asp Gly 675
680 685 Tyr Gln Leu Thr Gly His Ala Tyr Gln Met Cys Gln Asp Ala Glu
Asn 690 695 700 Gly Ile Trp Phe Lys Lys Ile Pro Leu Cys Lys Val Ile
His Cys His 705 710 715 720 Pro Pro Pro Val Ile Val Asn Gly Lys His
Thr Gly Met Met Ala Glu 725 730 735 Asn Phe Leu Tyr Gly Asn Glu Val
Ser Tyr Glu Cys Asp Gln Gly Phe 740 745 750 Tyr Leu Leu Gly Glu Lys
Lys Leu Gln Cys Arg Ser Asp Ser Lys Gly 755 760 765 His Gly Ser Trp
Ser Gly Pro Ser Pro Gln Cys Leu Arg Ser Pro Pro 770 775 780 Val Thr
Arg Cys Pro Asn Pro Glu Val Lys His Gly Tyr Lys Leu Asn 785 790 795
800 Lys Thr His Ser Ala Tyr Ser His Asn Asp Ile Val Tyr Val Asp Cys
805 810 815 Asn Pro Gly Phe Ile Met Asn Gly Ser Arg Val Ile Arg Cys
His Thr 820 825 830 Asp Asn Thr Trp Val Pro Gly Val Pro Thr Cys Ile
Lys Lys Ala Phe 835 840 845 Ile Gly Cys Pro Pro Pro Pro Lys Thr Pro
Asn Gly Asn His Thr Gly 850 855 860 Gly Asn Ile Ala Arg Phe Ser Pro
Gly Met Ser Ile Leu Tyr Ser Cys 865 870 875 880 Asp Gln Gly Tyr Leu
Leu Val Gly Glu Ala Leu Leu Leu Cys Thr His 885 890 895 Glu Gly Thr
Trp Ser Gln Pro Ala Pro His Cys Lys Glu Val Asn Cys 900 905 910 Ser
Ser Pro Ala Asp Met Asp Gly Ile Gln Lys Gly Leu Glu Pro Arg 915 920
925 Lys Met Tyr Gln Tyr Gly Ala Val Val Thr Leu Glu Cys Glu Asp Gly
930 935 940 Tyr Met Leu Glu Gly Ser Pro Gln Ser Gln Cys Gln Ser Asp
His Gln 945 950 955 960 Trp Asn Pro Pro Leu Ala Val Cys Arg Ser Arg
Ser Leu Ala Pro Val 965 970 975 Leu Cys Gly Ile Ala Ala Gly Leu Ile
Leu Leu Thr Phe Leu Ile Val 980 985 990 Ile Thr Leu Tyr Val Ile Ser
Lys His Arg Glu Arg Asn Tyr Tyr Thr 995 1000 1005 Asp Thr Ser Gln
Lys Glu Ala Phe His Leu Glu Ala Arg Glu Val Tyr 1010 1015 1020 Ser
Val Asp Pro Tyr Asn Pro Ala Ser 1025 1030 <210> SEQ ID NO 15
<211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
CD79B <400> SEQUENCE: 15 Met Ala Arg Leu Ala Leu Ser Pro Val
Pro Ser His Trp Met Val Ala 1 5 10 15 Leu Leu Leu Leu Leu Ser Ala
Glu Pro Val Pro Ala Ala Arg Ser Glu 20 25 30 Asp Arg Tyr Arg Asn
Pro Lys Gly Ser Ala Cys Ser Arg Ile Trp Gln 35 40 45 Ser Pro Arg
Phe Ile Ala Arg Lys Arg Gly Phe Thr Val Lys Met His 50 55 60 Cys
Tyr Met Asn Ser Ala Ser Gly Asn Val Ser Trp Leu Trp Lys Gln 65 70
75 80 Glu Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu Lys Gly Arg
Met 85 90 95 Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr Ile
Gln Gly Ile 100 105 110 Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln
Gln Lys Cys Asn Asn 115 120 125 Thr Ser Glu Val Tyr Gln Gly Cys Gly
Thr Glu Leu Arg Val Met Gly 130 135 140 Phe Ser Thr Leu Ala Gln Leu
Lys Gln Arg Asn Thr Leu Lys Asp Gly 145 150 155 160 Ile Ile Met Ile
Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val Pro 165 170 175 Ile Phe
Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly Met Glu Glu 180 185 190
Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu 195
200 205 Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp Ser Val Gly
Glu 210 215 220 His Pro Gly Gln Glu 225 <210> SEQ ID NO 16
<211> LENGTH: 508 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
FcRH2 <400> SEQUENCE: 16 Met Leu Leu Trp Ser Leu Leu Val Ile
Phe Asp Ala Val Thr Glu Gln 1 5 10 15 Ala Asp Ser Leu Thr Leu Val
Ala Pro Ser Ser Val Phe Glu Gly Asp 20 25 30 Ser Ile Val Leu Lys
Cys Gln Gly Glu Gln Asn Trp Lys Ile Gln Lys 35 40 45 Met Ala Tyr
His Lys Asp Asn Lys Glu Leu Ser Val Phe Lys Lys Phe 50 55 60 Ser
Asp Phe Leu Ile Gln Ser Ala Val Leu Ser Asp Ser Gly Asn Tyr 65 70
75 80 Phe Cys Ser Thr Lys Gly Gln Leu Phe Leu Trp Asp Lys Thr Ser
Asn 85 90 95 Ile Val Lys Ile Lys Val Gln Glu Leu Phe Gln Arg Pro
Val Leu Thr 100 105 110 Ala Ser Ser Phe Gln Pro Ile Glu Gly Gly Pro
Val Ser Leu Lys Cys 115 120 125 Glu Thr Arg Leu Ser Pro Gln Arg Leu
Asp Val Gln Leu Gln Phe Cys 130 135 140 Phe Phe Arg Glu Asn Gln Val
Leu Gly Ser Gly Trp Ser Ser Ser Pro 145 150 155 160 Glu Leu Gln Ile
Ser Ala Val Trp Ser Glu Asp Thr Gly Ser Tyr Trp 165 170 175 Cys Lys
Ala Glu Thr Val Thr His Arg Ile Arg Lys Gln Ser Leu Gln 180 185 190
Ser Gln Ile His Val Gln Arg Ile Pro Ile Ser Asn Val Ser Leu Glu 195
200 205 Ile Arg Ala Pro Gly Gly Gln Val Thr Glu Gly Gln Lys Leu Ile
Leu 210 215 220 Leu Cys Ser Val Ala Gly Gly Thr Gly Asn Val Thr Phe
Ser Trp Tyr 225 230 235 240 Arg Glu Ala Thr Gly Thr Ser Met Gly Lys
Lys Thr Gln Arg Ser Leu 245 250 255 Ser Ala Glu Leu Glu Ile Pro Ala
Val Lys Glu Ser Asp Ala Gly Lys 260 265 270 Tyr Tyr Cys Arg Ala Asp
Asn Gly His Val Pro Ile Gln Ser Lys Val 275 280 285 Val Asn Ile Pro
Val Arg Ile Pro Val Ser Arg Pro Val Leu Thr Leu 290 295 300 Arg Ser
Pro Gly Ala Gln Ala Ala Val Gly Asp Leu Leu Glu Leu His 305 310 315
320 Cys Glu Ala Leu Arg Gly Ser Pro Pro Ile Leu Tyr Gln Phe Tyr His
325 330 335 Glu Asp Val Thr Leu Gly Asn Ser Ser Ala Pro Ser Gly Gly
Gly Ala 340 345 350 Ser Phe Asn Leu Ser Leu Thr Ala Glu His Ser Gly
Asn Tyr Ser Cys 355 360 365 Glu Ala Asn Asn Gly Leu Gly Ala Gln Cys
Ser Glu Ala Val Pro Val 370 375 380 Ser Ile Ser Gly Pro Asp Gly Tyr
Arg Arg Asp Leu Met Thr Ala Gly 385 390 395 400 Val Leu Trp Gly Leu
Phe Gly Val Leu Gly Phe Thr Gly Val Ala Leu 405 410 415 Leu Leu Tyr
Ala Leu Phe His Lys Ile Ser Gly Glu Ser Ser Ala Thr 420 425 430 Asn
Glu Pro Arg Gly Ala Ser Arg Pro Asn Pro Gln Glu Phe Thr Tyr 435 440
445 Ser Ser Pro Thr Pro Asp Met Glu Glu Leu Gln Pro Val Tyr Val Asn
450 455 460 Val Gly Ser Val Asp Val Asp Val Val Tyr Ser Gln Val Trp
Ser Met 465 470 475 480 Gln Gln Pro Glu Ser Ser Ala Asn Ile Arg Thr
Leu Leu Glu Asn Lys 485 490 495 Asp Ser Gln Val Ile Tyr Ser Ser Val
Lys Lys Ser 500 505 <210> SEQ ID NO 17 <211> LENGTH:
1255 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: HER2
<400> SEQUENCE: 17 Met Glu Leu Ala Ala Leu Cys Arg Trp Gly
Leu Leu Leu Ala Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln
Val Cys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser
Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly
Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro
Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80
Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85
90 95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn
Tyr 100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn
Thr Thr Pro 115 120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu
Leu Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val
Leu Ile Gln Arg Asn Pro Gln 145 150 155 160 Leu Cys Tyr Gln Asp Thr
Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala
Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro
Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205
Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210
215 220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln
Cys 225 230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys
Leu Ala Cys Leu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu
His Cys Pro Ala Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu
Ser Met Pro Asn Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser
Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val
Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln 305 310 315 320 Glu
Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330
335 Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu
340 345 350 Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly
Cys Lys 355 360 365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser
Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro
Glu Gln Leu Gln Val Phe 385 390 395 400 Glu Thr Leu Glu Glu Ile Thr
Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp
Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile
Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly
Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455
460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val
465 470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu
Leu His Thr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu
Gly Leu Ala Cys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp
Gly Pro Gly Pro Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu
Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly
Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys 545 550 555 560 Leu Pro
Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575
Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580
585 590 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp
Leu 595 600 605 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly
Ala Cys Gln 610 615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val
Asp Leu Asp Asp Lys 625 630 635 640 Gly Cys Pro Ala Glu Gln Arg Ala
Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile Leu
Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile Lys
Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685 Arg Leu
Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700
Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 705
710 715 720 Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val
Tyr Lys 725 730 735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile
Pro Val Ala Ile 740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys
Ala Asn Lys Glu Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met Ala Gly
Val Gly Ser Pro Tyr Val Ser Arg 770 775 780 Leu Leu Gly Ile Cys Leu
Thr Ser Thr Val Gln Leu Val Thr Gln Leu 785 790 795 800 Met Pro Tyr
Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815 Leu
Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825
830 Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala
835 840 845 Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr
Asp Phe 850 855 860 Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu
Tyr His Ala Asp 865 870 875 880 Gly Gly Lys Val Pro Ile Lys Trp Met
Ala Leu Glu Ser Ile Leu Arg 885 890 895 Arg Arg Phe Thr His Gln Ser
Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910 Trp Glu Leu Met Thr
Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925 Arg Glu Ile
Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940 Pro
Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 945 950
955 960 Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu
Phe 965 970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile
Gln Asn Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr
Phe Tyr Arg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp Met Gly Asp
Leu Val Asp Ala Glu Glu Tyr Leu 1010 1015 1020 Val Pro Gln Gln Gly
Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly 1025 1030 1035 1040 Gly
Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly 1045
1050 1055 Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala
Pro Arg 1060 1065 1070 Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser
Asp Val Phe Asp Gly 1075 1080 1085 Asp Leu Gly Met Gly Ala Ala Lys
Gly Leu Gln Ser Leu Pro Thr His 1090 1095 1100 Asp Pro Ser Pro Leu
Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu 1105 1110 1115 1120 Pro
Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln 1125
1130 1135 Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro
Ser Pro 1140 1145 1150 Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala
Gly Ala Thr Leu Glu 1155 1160 1165 Arg Pro Lys Thr Leu Ser Pro Gly
Lys Asn Gly Val Val Lys Asp Val 1170 1175 1180 Phe Ala Phe Gly Gly
Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln 1185 1190 1195 1200 Gly
Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala 1205
1210 1215 Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg
Gly Ala 1220 1225 1230 Pro Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala
Glu Asn Pro Glu Tyr 1235 1240 1245 Leu Gly Leu Asp Val Pro Val 1250
1255 <210> SEQ ID NO 18 <211> LENGTH: 344 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: CEACAM6 <400> SEQUENCE: 18 Met
Gly Pro Pro Ser Ala Pro Pro Cys Arg Leu His Val Pro Trp Lys 1 5 10
15 Glu Val Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr
20 25 30 Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala
Glu Gly 35 40 45 Lys Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln
Asn Arg Ile Gly 50 55 60 Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp
Gly Asn Ser Leu Ile Val 65 70 75 80 Gly Tyr Val Ile Gly Thr Gln Gln
Ala Thr Pro Gly Pro Ala Tyr Ser 85 90 95 Gly Arg Glu Thr Ile Tyr
Pro Asn Ala Ser Leu Leu Ile Gln Asn Val 100 105 110 Thr Gln Asn Asp
Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp 115 120 125 Leu Val
Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu 130 135 140
Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys 145
150 155 160 Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr
Thr Tyr 165 170 175 Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser
Pro Arg Leu Gln 180 185 190 Leu Ser Asn Gly Asn Met Thr Leu Thr Leu
Leu Ser Val Lys Arg Asn 195 200 205 Asp Ala Gly Ser Tyr Glu Cys Glu
Ile Gln Asn Pro Ala Ser Ala Asn 210 215 220 Arg Ser Asp Pro Val Thr
Leu Asn Val Leu Tyr Gly Pro Asp Val Pro 225 230 235 240 Thr Ile Ser
Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn 245 250 255 Leu
Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe 260 265
270 Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn
275 280 285 Ile Thr Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His
Asn Ser 290 295 300 Ala Thr Gly Leu Asn Arg Thr Thr Val Thr Met Ile
Thr Val Ser Gly 305 310 315 320 Ser Ala Pro Val Leu Ser Ala Val Ala
Thr Val Gly Ile Thr Ile Gly 325 330 335 Val Leu Ala Arg Val Ala Leu
Ile 340 <210> SEQ ID NO 19 <211> LENGTH: 411
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: DPEP1
<400> SEQUENCE: 19 Met Trp Ser Gly Trp Trp Leu Trp Pro Leu
Val Ala Val Cys Thr Ala 1 5 10 15 Asp Phe Phe Arg Asp Glu Ala Glu
Arg Ile Met Arg Asp Ser Pro Val 20 25 30 Ile Asp Gly His Asn Asp
Leu Pro Trp Gln Leu Leu Asp Met Phe Asn 35 40 45 Asn Arg Leu Gln
Asp Glu Arg Ala Asn Leu Thr Thr Leu Ala Gly Thr 50 55 60 His Thr
Asn Ile Pro Lys Leu Arg Ala Gly Phe Val Gly Gly Gln Phe 65 70 75 80
Trp Ser Val Tyr Thr Pro Cys Asp Thr Gln Asn Lys Asp Ala Val Arg 85
90 95 Arg Thr Leu Glu Gln Met Asp Val Val His Arg Met Cys Arg Met
Tyr 100 105 110 Pro Glu Thr Phe Leu Tyr Val Thr Ser Ser Ala Gly Ile
Arg Gln Ala 115 120 125 Phe Arg Glu Gly Lys Val Ala Ser Leu Ile Gly
Val Glu Gly Gly His 130 135 140 Ser Ile Asp Ser Ser Leu Gly Val Leu
Arg Ala Leu Tyr Gln Leu Gly 145 150 155 160 Met Arg Tyr Leu Thr Leu
Thr His Ser Cys Asn Thr Pro Trp Ala Asp 165 170 175 Asn Trp Leu Val
Asp Thr Gly Asp Ser Glu Pro Gln Ser Gln Gly Leu 180 185 190 Ser Pro
Phe Gly Gln Arg Val Val Lys Glu Leu Asn Arg Leu Gly Val 195 200 205
Leu Ile Asp Leu Ala His Val Ser Val Ala Thr Met Lys Ala Thr Leu 210
215 220 Gln Leu Ser Arg Ala Pro Val Ile Phe Ser His Ser Ser Ala Tyr
Ser 225 230 235 240 Val Cys Ala Ser Arg Arg Asn Val Pro Asp Asp Val
Leu Arg Leu Val 245 250 255 Lys Gln Thr Asp Ser Leu Val Met Val Asn
Phe Tyr Asn Asn Tyr Ile 260 265 270 Ser Cys Thr Asn Lys Ala Asn Leu
Ser Gln Val Ala Asp His Leu Asp 275 280 285 His Ile Lys Glu Val Ala
Gly Ala Arg Ala Val Gly Phe Gly Gly Asp 290 295 300 Phe Asp Gly Val
Pro Arg Val Pro Glu Gly Leu Glu Asp Val Ser Lys 305 310 315 320 Tyr
Pro Asp Leu Ile Ala Glu Leu Leu Arg Arg Asn Trp Thr Glu Ala 325 330
335 Glu Val Lys Gly Ala Leu Ala Asp Asn Leu Leu Arg Val Phe Glu Ala
340 345 350 Val Glu Gln Ala Ser Asn Leu Thr Gln Ala Pro Glu Glu Glu
Pro Ile 355 360 365 Pro Leu Asp Gln Leu Gly Gly Ser Cys Arg Thr His
Tyr Gly Tyr Ser 370 375 380 Ser Gly Ala Ser Ser Leu His Arg His Trp
Gly Leu Leu Leu Ala Ser 385 390 395 400 Leu Ala Pro Leu Val Leu Cys
Leu Ser Leu Leu 405 410 <210> SEQ ID NO 20 <211>
LENGTH: 553 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <223> OTHER INFORMATION: IL20Ra
<400> SEQUENCE: 20 Met Arg Ala Pro Gly Arg Pro Ala Leu Arg
Pro Leu Pro Leu Pro Pro 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala Ala
Pro Trp Gly Arg Ala Val Pro Cys 20 25 30 Val Ser Gly Gly Leu Pro
Lys Pro Ala Asn Ile Thr Phe Leu Ser Ile 35 40 45 Asn Met Lys Asn
Val Leu Gln Trp Thr Pro Pro Glu Gly Leu Gln Gly 50 55 60 Val Lys
Val Thr Tyr Thr Val Gln Tyr Phe Ile Tyr Gly Gln Lys Lys 65 70 75 80
Trp Leu Asn Lys Ser Glu Cys Arg Asn Ile Asn Arg Thr Tyr Cys Asp 85
90 95 Leu Ser Ala Glu Thr Ser Asp Tyr Glu His Gln Tyr Tyr Ala Lys
Val 100 105 110 Lys Ala Ile Trp Gly Thr Lys Cys Ser Lys Trp Ala Glu
Ser Gly Arg 115 120 125 Phe Tyr Pro Phe Leu Glu Thr Gln Ile Gly Pro
Pro Glu Val Ala Leu 130 135 140 Thr Thr Asp Glu Lys Ser Ile Ser Val
Val Leu Thr Ala Pro Glu Lys 145 150 155 160 Trp Lys Arg Asn Pro Glu
Asp Leu Pro Val Ser Met Gln Gln Ile Tyr 165 170 175 Ser Asn Leu Lys
Tyr Asn Val Ser Val Leu Asn Thr Lys Ser Asn Arg 180 185 190 Thr Trp
Ser Gln Cys Val Thr Asn His Thr Leu Val Leu Thr Trp Leu 195 200 205
Glu Pro Asn Thr Leu Tyr Cys Val His Val Glu Ser Phe Val Pro Gly 210
215 220 Pro Pro Arg Arg Ala Gln Pro Ser Glu Lys Gln Cys Ala Arg Thr
Leu 225 230 235 240 Lys Asp Gln Ser Ser Glu Phe Lys Ala Lys Ile Ile
Phe Trp Tyr Val 245 250 255 Leu Pro Ile Ser Ile Thr Val Phe Leu Phe
Ser Val Met Gly Tyr Ser 260 265 270 Ile Tyr Arg Tyr Ile His Val Gly
Lys Glu Lys His Pro Ala Asn Leu 275 280 285 Ile Leu Ile Tyr Gly Asn
Glu Phe Asp Lys Arg Phe Phe Val Pro Ala 290 295 300 Glu Lys Ile Val
Ile Asn Phe Ile Thr Leu Asn Ile Ser Asp Asp Ser 305 310 315 320 Lys
Ile Ser His Gln Asp Met Ser Leu Leu Gly Lys Ser Ser Asp Val 325 330
335 Ser Ser Leu Asn Asp Pro Gln Pro Ser Gly Asn Leu Arg Pro Pro Gln
340 345 350 Glu Glu Glu Glu Val Lys His Leu Gly Tyr Ala Ser His Leu
Met Glu 355 360 365 Ile Phe Cys Asp Ser Glu Glu Asn Thr Glu Gly Thr
Ser Phe Thr Gln 370 375 380 Gln Glu Ser Leu Ser Arg Thr Ile Pro Pro
Asp Lys Thr Val Ile Glu 385 390 395 400 Tyr Glu Tyr Asp Val Arg Thr
Thr Asp Ile Cys Ala Gly Pro Glu Glu 405 410 415 Gln Glu Leu Ser Leu
Gln Glu Glu Val Ser Thr Gln Gly Thr Leu Leu 420 425 430 Glu Ser Gln
Ala Ala Leu Ala Val Leu Gly Pro Gln Thr Leu Gln Tyr 435 440 445 Ser
Tyr Thr Pro Gln Leu Gln Asp Leu Asp Pro Leu Ala Gln Glu His 450 455
460 Thr Asp Ser Glu Glu Gly Pro Glu Glu Glu Pro Ser Thr Thr Leu Val
465 470 475 480 Asp Trp Asp Pro Gln Thr Gly Arg Leu Cys Ile Pro Ser
Leu Ser Ser 485 490 495 Phe Asp Gln Asp Ser Glu Gly Cys Glu Pro Ser
Glu Gly Asp Gly Leu 500 505 510 Gly Glu Glu Gly Leu Leu Ser Arg Leu
Tyr Glu Glu Pro Ala Pro Asp 515 520 525 Arg Pro Pro Gly Glu Asn Glu
Thr Tyr Leu Met Gln Phe Met Glu Glu 530 535 540 Trp Gly Leu Tyr Val
Gln Met Glu Asn 545 550 <210> SEQ ID NO 21 <211>
LENGTH: 911 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <223> OTHER INFORMATION: BCAN
<400> SEQUENCE: 21 Met Ala Gln Leu Phe Leu Pro Leu Leu Ala
Ala Leu Val Leu Ala Gln 1 5 10 15 Ala Pro Ala Ala Leu Ala Asp Val
Leu Glu Gly Asp Ser Ser Glu Asp 20 25 30 Arg Ala Phe Arg Val Arg
Ile Ala Gly Asp Ala Pro Leu Gln Gly Val 35 40 45 Leu Gly Gly Ala
Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro 50 55 60 Pro Pro
Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr 65 70 75 80
Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Val 85
90 95 Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro
Ala 100 105 110 Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala Leu Ser
Glu Leu Arg 115 120 125 Pro Asn Asp Ser Gly Ile Tyr Arg Cys Glu Val
Gln His Gly Ile Asp 130 135 140 Asp Ser Ser Asp Ala Val Glu Val Lys
Val Lys Gly Val Val Phe Leu 145 150 155 160 Tyr Arg Glu Gly Ser Ala
Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln 165 170 175 Glu Ala Cys Ala
Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu 180 185 190 Tyr Ala
Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu 195 200 205
Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala Cys 210
215 220 Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly Val
Val 225 230 235 240 Asp Pro Asp Asp Leu Tyr Asp Val Tyr Cys Tyr Ala
Glu Asp Leu Asn 245 250 255 Gly Glu Leu Phe Leu Gly Asp Pro Pro Glu
Lys Leu Thr Leu Glu Glu 260 265 270 Ala Arg Ala Tyr Cys Gln Glu Arg
Gly Ala Glu Ile Ala Thr Thr Gly 275 280 285 Gln Leu Tyr Ala Ala Trp
Asp Gly Gly Leu Asp His Cys Ser Pro Gly 290 295 300 Trp Leu Ala Asp
Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln 305 310 315 320 Arg
Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro 325 330
335 Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val Tyr Cys
340 345 350 Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser
Asn Pro 355 360 365 Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu Ala Ile
Val Thr Val Thr 370 375 380 Glu Thr Leu Glu Glu Leu Gln Leu Pro Gln
Glu Ala Thr Glu Ser Glu 385 390 395 400 Ser Arg Gly Ala Ile Tyr Ser
Ile Pro Ile Met Glu Asp Gly Gly Gly 405 410 415 Gly Ser Ser Thr Pro
Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu 420 425 430 Glu Phe Glu
Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu 435 440 445 Glu
Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu 450 455
460 Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala Leu Trp
465 470 475 480 Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro Glu Ala
Ser Leu Pro 485 490 495 Thr Glu Pro Ala Ala Gln Glu Lys Ser Leu Ser
Gln Ala Pro Ala Arg 500 505 510 Ala Val Leu Gln Pro Gly Ala Ser Pro
Leu Pro Asp Gly Glu Ser Glu 515 520 525 Ala Ser Arg Pro Pro Arg Val
His Gly Pro Pro Thr Glu Thr Leu Pro 530 535 540 Thr Pro Arg Glu Arg
Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val 545 550 555 560 Glu Ala
Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly 565 570 575
Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala Pro 580
585 590 Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg Glu Leu
Glu 595 600 605 Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala Pro Ala
Gly Thr Ser 610 615 620 Val Gln Ala Gln Pro Val Leu Pro Thr Asp Ser
Ala Ser Arg Gly Gly 625 630 635 640 Val Ala Val Val Pro Ala Ser Gly
Asp Cys Val Pro Ser Pro Cys His 645 650 655 Asn Gly Gly Thr Cys Leu
Glu Glu Glu Glu Gly Val Arg Cys Leu Cys 660 665 670 Leu Pro Gly Tyr
Gly Gly Asp Leu Cys Asp Val Gly Leu Arg Phe Cys 675 680 685 Asn Pro
Gly Trp Asp Ala Phe Gln Gly Ala Cys Tyr Lys His Phe Ser 690 695 700
Thr Arg Arg Ser Trp Glu Glu Ala Glu Thr Gln Cys Arg Met Tyr Gly 705
710 715 720 Ala His Leu Ala Ser Ile Ser Thr Pro Glu Glu Gln Asp Phe
Ile Asn 725 730 735 Asn Arg Tyr Arg Glu Tyr Gln Trp Ile Gly Leu Asn
Asp Arg Thr Ile 740 745 750 Glu Gly Asp Phe Leu Trp Ser Asp Gly Val
Pro Leu Leu Tyr Glu Asn 755 760 765 Trp Asn Pro Gly Gln Pro Asp Ser
Tyr Phe Leu Ser Gly Glu Asn Cys 770 775 780 Val Val Met Val Trp His
Asp Gln Gly Gln Trp Ser Asp Val Pro Cys 785 790 795 800 Asn Tyr His
Leu Ser Tyr Thr Cys Lys Met Gly Leu Val Ser Cys Gly 805 810 815 Pro
Pro Pro Glu Leu Pro Leu Ala Gln Val Phe Gly Arg Pro Arg Leu 820 825
830 Arg Tyr Glu Val Asp Thr Val Leu Arg Tyr Arg Cys Arg Glu Gly Leu
835 840 845 Ala Gln Arg Asn Leu Pro Leu Ile Arg Cys Gln Glu Asn Gly
Arg Trp 850 855 860 Glu Ala Pro Gln Ile Ser Cys Val Pro Arg Arg Pro
Ala Arg Ala Leu 865 870 875 880 His Pro Glu Glu Asp Pro Glu Gly Arg
Gln Gly Arg Leu Leu Gly Arg 885 890 895 Trp Lys Ala Leu Leu Ile Pro
Pro Ser Ser Pro Met Pro Gly Pro 900 905 910 <210> SEQ ID NO
22 <211> LENGTH: 987 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: EphB2R <400> SEQUENCE: 22 Met Ala Leu Arg Arg
Leu Gly Ala Ala Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Ala Ala Val
Glu Glu Thr Leu Met Asp Ser Thr Thr Ala Thr Ala Glu 20 25 30 Leu
Gly Trp Met Val His Pro Pro Ser Gly Trp Glu Glu Val Ser Gly 35 40
45 Tyr Asp Glu Asn Met Asn Thr Ile Arg Thr Tyr Gln Val Cys Asn Val
50 55 60 Phe Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Lys Phe Ile
Arg Arg 65 70 75 80 Arg Gly Ala His Arg Ile His Val Glu Met Lys Phe
Ser Val Arg Asp 85 90 95 Cys Ser Ser Ile Pro Ser Val Pro Gly Ser
Cys Lys Glu Thr Phe Asn 100 105 110 Leu Tyr Tyr Tyr Glu Ala Asp Phe
Asp Ser Ala Thr Lys Thr Phe Pro 115 120 125 Asn Trp Met Glu Asn Pro
Trp Val Lys Val Asp Thr Ile Ala Ala Asp 130 135 140 Glu Ser Phe Ser
Gln Val Asp Leu Gly Gly Arg Val Met Lys Ile Asn 145 150 155 160 Thr
Glu Val Arg Ser Phe Gly Pro Val Ser Arg Ser Gly Phe Tyr Leu 165 170
175 Ala Phe Gln Asp Tyr Gly Gly Cys Met Ser Leu Ile Ala Val Arg Val
180 185 190 Phe Tyr Arg Lys Cys Pro Arg Ile Ile Gln Asn Gly Ala Ile
Phe Gln 195 200 205 Glu Thr Leu Ser Gly Ala Glu Ser Thr Ser Leu Val
Ala Ala Arg Gly 210 215 220 Ser Cys Ile Ala Asn Ala Glu Glu Val Asp
Val Pro Ile Lys Leu Tyr 225 230 235 240 Cys Asn Gly Asp Gly Glu Trp
Leu Val Pro Ile Gly Arg Cys Met Cys 245 250 255 Lys Ala Gly Phe Glu
Ala Val Glu Asn Gly Thr Val Cys Arg Gly Cys 260 265 270 Pro Ser Gly
Thr Phe Lys Ala Asn Gln Gly Asp Glu Ala Cys Thr His 275 280 285 Cys
Pro Ile Asn Ser Arg Thr Thr Ser Glu Gly Ala Thr Asn Cys Val 290 295
300 Cys Arg Asn Gly Tyr Tyr Arg Ala Asp Leu Asp Pro Leu Asp Met Pro
305 310 315 320 Cys Thr Thr Ile Pro Ser Ala Pro Gln Ala Val Ile Ser
Ser Val Asn 325 330 335 Glu Thr Ser Leu Met Leu Glu Trp Thr Pro Pro
Arg Asp Ser Gly Gly 340 345 350 Arg Glu Asp Leu Val Tyr Asn Ile Ile
Cys Lys Ser Cys Gly Ser Gly 355 360 365 Arg Gly Ala Cys Thr Arg Cys
Gly Asp Asn Val Gln Tyr Ala Pro Arg 370 375 380 Gln Leu Gly Leu Thr
Glu Pro Arg Ile Tyr Ile Ser Asp Leu Leu Ala 385 390 395 400 His Thr
Gln Tyr Thr Phe Glu Ile Gln Ala Val Asn Gly Val Thr Asp 405 410 415
Gln Ser Pro Phe Ser Pro Gln Phe Ala Ser Val Asn Ile Thr Thr Asn 420
425 430 Gln Ala Ala Pro Ser Ala Val Ser Ile Met His Gln Val Ser Arg
Thr 435 440 445 Val Asp Ser Ile Thr Leu Ser Trp Ser Gln Pro Asp Gln
Pro Asn Gly 450 455 460 Val Ile Leu Asp Tyr Glu Leu Gln Tyr Tyr Glu
Lys Glu Leu Ser Glu 465 470 475 480 Tyr Asn Ala Thr Ala Ile Lys Ser
Pro Thr Asn Thr Val Thr Val Gln 485 490 495 Gly Leu Lys Ala Gly Ala
Ile Tyr Val Phe Gln Val Arg Ala Arg Thr 500 505 510 Val Ala Gly Tyr
Gly Arg Tyr Ser Gly Lys Met Tyr Phe Gln Thr Met 515 520 525 Thr Glu
Ala Glu Tyr Gln Thr Ser Ile Gln Glu Lys Leu Pro Leu Ile 530 535 540
Ile Gly Ser Ser Ala Ala Gly Leu Val Phe Leu Ile Ala Val Val Val 545
550 555 560 Ile Ala Ile Val Cys Asn Arg Arg Arg Gly Phe Glu Arg Ala
Asp Ser 565 570 575 Glu Tyr Thr Asp Lys Leu Gln His Tyr Thr Ser Gly
His Met Thr Pro 580 585 590 Gly Met Lys Ile Tyr Ile Asp Pro Phe Thr
Tyr Glu Asp Pro Asn Glu 595 600 605 Ala Val Arg Glu Phe Ala Lys Glu
Ile Asp Ile Ser Cys Val Lys Ile 610 615 620 Glu Gln Val Ile Gly Ala
Gly Glu Phe Gly Glu Val Cys Ser Gly His 625 630 635 640 Leu Lys Leu
Pro Gly Lys Arg Glu Ile Phe Val Ala Ile Lys Thr Leu 645 650 655 Lys
Ser Gly Tyr Thr Glu Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala 660 665
670 Ser Ile Met Gly Gln Phe Asp His Pro Asn Val Ile His Leu Glu Gly
675 680 685 Val Val Thr Lys Ser Thr Pro Val Met Ile Ile Thr Glu Phe
Met Glu 690 695 700 Asn Gly Ser Leu Asp Ser Phe Leu Arg Gln Asn Asp
Gly Gln Phe Thr 705 710 715 720 Val Ile Gln Leu Val Gly Met Leu Arg
Gly Ile Ala Ala Gly Met Lys 725 730 735 Tyr Leu Ala Asp Met Asn Tyr
Val His Arg Asp Leu Ala Ala Arg Asn 740 745 750 Ile Leu Val Asn Ser
Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu 755 760 765 Ser Arg Phe
Leu Glu Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser Ala 770 775 780 Leu
Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Gln 785 790
795 800 Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile
Val 805 810 815 Met Trp Glu Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp
Asp Met Thr 820 825 830 Asn Gln Asp Val Ile Asn Ala Ile Glu Gln Asp
Tyr Arg Leu Pro Pro 835 840 845 Pro Met Asp Cys Pro Ser Ala Leu His
Gln Leu Met Leu Asp Cys Trp 850 855 860 Gln Lys Asp Arg Asn His Arg
Pro Lys Phe Gly Gln Ile Val Asn Thr 865 870 875 880 Leu Asp Lys Met
Ile Arg Asn Pro Asn Ser Leu Lys Ala Met Ala Pro 885 890 895 Leu Ser
Ser Gly Ile Asn Leu Pro Leu Leu Asp Arg Thr Ile Pro Asp 900 905 910
Tyr Thr Ser Phe Asn Thr Val Asp Glu Trp Leu Glu Ala Ile Lys Met 915
920 925 Gly Gln Tyr Lys Glu Ser Phe Ala Asn Ala Gly Phe Thr Ser Phe
Asp 930 935 940 Val Val Ser Gln Met Met Met Glu Asp Ile Leu Arg Val
Gly Val Thr 945 950 955 960 Leu Ala Gly His Gln Lys Lys Ile Leu Asn
Ser Ile Gln Val Met Arg 965 970 975 Ala Gln Met Asn Gln Ile Gln Ser
Val Glu Val 980 985 <210> SEQ ID NO 23 <211> LENGTH:
282 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: ASLG659
<400> SEQUENCE: 23 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp
Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala
Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr
Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly
Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp
Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80
His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85
90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly
Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala
Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Lys
Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro
Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu
Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val
Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val
Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205
Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210
215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys
Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln
Leu Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe
Ala Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met
Leu Lys 275 280 <210> SEQ ID NO 24 <211> LENGTH: 123
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: PSCA
<400> SEQUENCE: 24 Met Lys Ala Val Leu Leu Ala Leu Leu Met
Ala Gly Leu Ala Leu Gln 1 5 10 15 Pro Gly Thr Ala Leu Leu Cys Tyr
Ser Cys Lys Ala Gln Val Ser Asn 20 25 30 Glu Asp Cys Leu Gln Val
Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys 35 40 45 Trp Thr Ala Arg
Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys 50 55 60 Gly Cys
Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly 65 70 75 80
Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly 85
90 95 Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro
Ala 100 105 110 Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu 115 120
<210> SEQ ID NO 25 <211> LENGTH: 236 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: GEDA <400> SEQUENCE: 25 Met
Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Met Leu 1 5 10
15 Pro Ala Gln Glu Ala Ala Lys Leu Tyr His Thr Asn Tyr Val Arg Asn
20 25 30 Ser Arg Ala Ile Gly Val Leu Trp Ala Ile Phe Thr Ile Cys
Phe Ala 35 40 45 Ile Val Asn Val Val Cys Phe Ile Gln Pro Tyr Trp
Ile Gly Asp Gly 50 55 60 Val Asp Thr Pro Gln Ala Gly Tyr Phe Gly
Leu Phe His Tyr Cys Ile 65 70 75 80 Gly Asn Gly Phe Ser Arg Glu Leu
Thr Cys Arg Gly Ser Phe Thr Asp 85 90 95 Phe Ser Thr Leu Pro Ser
Gly Ala Phe Lys Ala Ala Ser Phe Phe Ile 100 105 110 Gly Leu Ser Met
Met Leu Ile Ile Ala Cys Ile Ile Cys Phe Thr Leu 115 120 125 Phe Phe
Phe Cys Asn Thr Ala Thr Val Tyr Lys Ile Cys Ala Trp Met 130 135 140
Gln Leu Thr Ser Ala Ala Cys Leu Val Leu Gly Cys Met Ile Phe Pro 145
150 155 160 Asp Gly Trp Asp Ser Asp Glu Val Lys Arg Met Cys Gly Glu
Lys Thr 165 170 175 Asp Lys Tyr Thr Leu Gly Ala Cys Ser Val Arg Trp
Ala Tyr Ile Leu 180 185 190 Ala Ile Ile Gly Ile Leu Asp Ala Leu Ile
Leu Ser Phe Leu Ala Phe 195 200 205 Val Leu Gly Asn Arg Gln Asp Ser
Leu Met Ala Glu Glu Leu Lys Ala 210 215 220 Glu Asn Lys Val Leu Leu
Ser Gln Tyr Ser Leu Glu 225 230 235 <210> SEQ ID NO 26
<211> LENGTH: 184 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
BAFF-R <400> SEQUENCE: 26 Met Arg Arg Gly Pro Arg Ser Leu Arg
Gly Arg Asp Ala Pro Ala Pro 1 5 10 15 Thr Pro Cys Val Pro Ala Glu
Cys Phe Asp Leu Leu Val Arg His Cys 20 25 30 Val Ala Cys Gly Leu
Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala 35 40 45 Ser Ser Pro
Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val Gly 50 55 60 Ala
Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu Phe Gly 65 70
75 80 Ala Pro Ala Leu Leu Gly Leu Ala Leu Val Leu Ala Leu Val Leu
Val 85 90 95 Gly Leu Val Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg
Gly Ala Ser 100 105 110 Ser Ala Glu Ala Pro Asp Gly Asp Lys Asp Ala
Pro Glu Pro Leu Asp 115 120 125 Lys Val Ile Ile Leu Ser Pro Gly Ile
Ser Asp Ala Thr Ala Pro Ala 130 135 140 Trp Pro Pro Pro Gly Glu Asp
Pro Gly Thr Thr Pro Pro Gly His Ser 145 150 155 160 Val Pro Val Pro
Ala Thr Glu Leu Gly Ser Thr Glu Leu Val Thr Thr 165 170 175 Lys Thr
Ala Gly Pro Glu Gln Gln 180 <210> SEQ ID NO 27 <211>
LENGTH: 847 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <223> OTHER INFORMATION: CD22
<400> SEQUENCE: 27 Met His Leu Leu Gly Pro Trp Leu Leu Leu
Leu Val Leu Glu Tyr Leu 1 5 10 15 Ala Phe Ser Asp Ser Ser Lys Trp
Val Phe Glu His Pro Glu Thr Leu 20 25 30 Tyr Ala Trp Glu Gly Ala
Cys Val Trp Ile Pro Cys Thr Tyr Arg Ala 35 40 45 Leu Asp Gly Asp
Leu Glu Ser Phe Ile Leu Phe His Asn Pro Glu Tyr 50 55 60 Asn Lys
Asn Thr Ser Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr 65 70 75 80
Lys Asp Gly Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly 85
90 95 Asp Lys Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu
Asn 100 105 110 Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr
Glu Lys Trp 115 120 125 Met Glu Arg Ile His Leu Asn Val Ser Glu Arg
Pro Phe Pro Pro His 130 135 140 Ile Gln Leu Pro Pro Glu Ile Gln Glu
Ser Gln Glu Val Thr Leu Thr 145 150 155 160 Cys Leu Leu Asn Phe Ser
Cys Tyr Gly Tyr Pro Ile Gln Leu Gln Trp 165 170 175 Leu Leu Glu Gly
Val Pro Met Arg Gln Ala Ala Val Thr Ser Thr Ser 180 185 190 Leu Thr
Ile Lys Ser Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro 195 200 205
Gln Trp Ser His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala 210
215 220 Asp Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys
His 225 230 235 240 Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp
Ala Ile Val Arg 245 250 255 Glu Gly Asp Ser Val Thr Met Thr Cys Glu
Val Ser Ser Ser Asn Pro 260 265 270 Glu Tyr Thr Thr Val Ser Trp Leu
Lys Asp Gly Thr Ser Leu Lys Lys 275 280 285 Gln Asn Thr Phe Thr Leu
Asn Leu Arg Glu Val Thr Lys Asp Gln Ser 290 295 300 Gly Lys Tyr Cys
Cys Gln Val Ser Asn Asp Val Gly Pro Gly Arg Ser 305 310 315 320 Glu
Glu Val Phe Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val 325 330
335 Gln Ile Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu
340 345 350 Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp
Tyr His 355 360 365 Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys
Val His Ile Pro 370 375 380 Lys Ile Leu Pro Trp His Ala Gly Thr Tyr
Ser Cys Val Ala Glu Asn 385 390 395 400 Ile Leu Gly Thr Gly Gln Arg
Gly Pro Gly Ala Glu Leu Asp Val Gln 405 410 415 Tyr Pro Pro Lys Lys
Val Thr Thr Val Ile Gln Asn Pro Met Pro Ile 420 425 430 Arg Glu Gly
Asp Thr Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn 435 440 445 Pro
Ser Val Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu 450 455
460 Pro Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr
465 470 475 480 Thr Ile Ala Cys Ala Arg Cys Asn Ser Trp Cys Ser Trp
Ala Ser Pro 485 490 495 Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp
Val Arg Val Arg Lys 500 505 510 Ile Lys Pro Leu Ser Glu Ile His Ser
Gly Asn Ser Val Ser Leu Gln 515 520 525 Cys Asp Phe Ser Ser Ser His
Pro Lys Glu Val Gln Phe Phe Trp Glu 530 535 540 Lys Asn Gly Arg Leu
Leu Gly Lys Glu Ser Gln Leu Asn Phe Asp Ser 545 550 555 560 Ile Ser
Pro Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser 565 570 575
Ile Gly Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala 580
585 590 Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met
Glu 595 600 605 Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn
Pro Pro Val 610 615 620 Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln
Ser Leu Pro His His 625 630 635 640 Ser Gln Lys Leu Arg Leu Glu Pro
Val Lys Val Gln His Ser Gly Ala 645 650 655 Tyr Trp Cys Gln Gly Thr
Asn Ser Val Gly Lys Gly Arg Ser Pro Leu 660 665 670 Ser Thr Leu Thr
Val Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Val 675 680 685 Ala Val
Gly Leu Gly Ser Cys Leu Ala Ile Leu Ile Leu Ala Ile Cys 690 695 700
Gly Leu Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly 705
710 715 720 Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn
Lys Lys 725 730 735 Val Arg Arg Ala Pro Leu Ser Glu Gly Pro His Ser
Leu Gly Cys Tyr 740 745 750 Asn Pro Met Met Glu Asp Gly Ile Ser Tyr
Thr Thr Leu Arg Phe Pro 755 760 765 Glu Met Asn Ile Pro Arg Thr Gly
Asp Ala Glu Ser Ser Glu Met Gln 770 775 780 Arg Pro Pro Arg Thr Cys
Asp Asp Thr Val Thr Tyr Ser Ala Leu His 785 790 795 800 Lys Arg Gln
Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu 805 810 815 Asp
Glu Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu 820 825
830 Arg Pro Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His 835
840 845 <210> SEQ ID NO 28 <211> LENGTH: 226
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CD79A
<400> SEQUENCE: 28 Met Pro Gly Gly Pro Gly Val Leu Gln Ala
Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val
Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val
Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe
Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp
Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80
Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85
90 95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly
Asn 100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val
Arg Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly
Thr Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu
Phe Cys Ala Val Val Pro Gly 145 150 155 160 Thr Leu Leu Leu Phe Arg
Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp
Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp
Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205
Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210
215 220 Lys Pro 225 <210> SEQ ID NO 29 <211> LENGTH:
372 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CXCR5
<400> SEQUENCE: 29 Met Asn Tyr Pro Leu Thr Leu Glu Met Asp
Leu Glu Asn Leu Glu Asp 1 5 10 15 Leu Phe Trp Glu Leu Asp Arg Leu
Asp Asn Tyr Asn Asp Thr Ser Leu 20 25 30 Val Glu Asn His Leu Cys
Pro Ala Thr Glu Gly Pro Leu Met Ala Ser 35 40 45 Phe Lys Ala Val
Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu 50 55 60 Gly Val
Ile Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg 65 70 75 80
Gln Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala 85
90 95 Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly
Ser 100 105 110 Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val
Ile Ala Leu 115 120 125 His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu
Leu Ala Cys Ile Ala 130 135 140 Val Asp Arg Tyr Leu Ala Ile Val His
Ala Val His Ala Tyr Arg His 145 150 155 160 Arg Arg Leu Leu Ser Ile
His Ile Thr Cys Gly Thr Ile Trp Leu Val 165 170 175 Gly Phe Leu Leu
Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln 180 185 190 Gly His
His Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn 195 200 205
Gln Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val 210
215 220 Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val
Gly 225 230 235 240 Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro
Gln Arg Gln Lys 245 250 255 Ala Val Arg Val Ala Ile Leu Val Thr Ser
Ile Phe Phe Leu Cys Trp 260 265 270 Ser Pro Tyr His Ile Val Ile Phe
Leu Asp Thr Leu Ala Arg Leu Lys 275 280 285 Ala Val Asp Asn Thr Cys
Lys Leu Asn Gly Ser Leu Pro Val Ala Ile 290 295 300 Thr Met Cys Glu
Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met 305 310 315 320 Leu
Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu 325 330
335 Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe
340 345 350 Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala
Thr Ser 355 360 365 Leu Thr Thr Phe 370 <210> SEQ ID NO 30
<211> LENGTH: 273 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
HLA-DOB <400> SEQUENCE: 30 Met Gly Ser Gly Trp Val Pro Trp
Val Val Ala Leu Leu Val Asn Leu 1 5 10 15 Thr Arg Leu Asp Ser Ser
Met Thr Gln Gly Thr Asp Ser Pro Glu Asp 20 25 30 Phe Val Ile Gln
Ala Lys Ala Asp Cys Tyr Phe Thr Asn Gly Thr Glu 35 40 45 Lys Val
Gln Phe Val Val Arg Phe Ile Phe Asn Leu Glu Glu Tyr Val 50 55 60
Arg Phe Asp Ser Asp Val Gly Met Phe Val Ala Leu Thr Lys Leu Gly 65
70 75 80 Gln Pro Asp Ala Glu Gln Trp Asn Ser Arg Leu Asp Leu Leu
Glu Arg 85 90 95 Ser Arg Gln Ala Val Asp Gly Val Cys Arg His Asn
Tyr Arg Leu Gly 100 105 110 Ala Pro Phe Thr Val Gly Arg Lys Val Gln
Pro Glu Val Thr Val Tyr 115 120 125 Pro Glu Arg Thr Pro Leu Leu His
Gln His Asn Leu Leu His Cys Ser 130 135 140 Val Thr Gly Phe Tyr Pro
Gly Asp Ile Lys Ile Lys Trp Phe Leu Asn 145 150 155 160 Gly Gln Glu
Glu Arg Ala Gly Val Met Ser Thr Gly Pro Ile Arg Asn 165 170 175 Gly
Asp Trp Thr Phe Gln Thr Val Val Met Leu Glu Met Thr Pro Glu 180 185
190 Leu Gly His Val Tyr Thr Cys Leu Val Asp His Ser Ser Leu Leu Ser
195 200 205 Pro Val Ser Val Glu Trp Arg Ala Gln Ser Glu Tyr Ser Trp
Arg Lys 210 215 220 Met Leu Ser Gly Ile Ala Ala Phe Leu Leu Gly Leu
Ile Phe Leu Leu 225 230 235 240 Val Gly Ile Val Ile Gln Leu Arg Ala
Gln Lys Gly Tyr Val Arg Thr 245 250 255 Gln Met Ser Gly Asn Glu Val
Ser Arg Ala Val Leu Leu Pro Gln Ser 260 265 270 Cys <210> SEQ
ID NO 31 <211> LENGTH: 422 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Purinergic receptor P2X ligand-gated ion channel 5
<400> SEQUENCE: 31 Met Gly Gln Ala Gly Cys Lys Gly Leu Cys
Leu Ser Leu Phe Asp Tyr 1 5 10 15 Lys Thr Glu Lys Tyr Val Ile Ala
Lys Asn Lys Lys Val Gly Leu Leu 20 25 30 Tyr Arg Leu Leu Gln Ala
Ser Ile Leu Ala Tyr Leu Val Val Trp Val 35 40 45 Phe Leu Ile Lys
Lys Gly Tyr Gln Asp Val Asp Thr Ser Leu Gln Ser 50 55 60 Ala Val
Ile Thr Lys Val Lys Gly Val Ala Phe Thr Asn Thr Ser Asp 65 70 75 80
Leu Gly Gln Arg Ile Trp Asp Val Ala Asp Tyr Val Ile Pro Ala Gln 85
90 95 Gly Glu Asn Val Phe Phe Val Val Thr Asn Leu Ile Val Thr Pro
Asn 100 105 110 Gln Arg Gln Asn Val Cys Ala Glu Asn Glu Gly Ile Pro
Asp Gly Ala 115 120 125 Cys Ser Lys Asp Ser Asp Cys His Ala Gly Glu
Ala Val Thr Ala Gly 130 135 140 Asn Gly Val Lys Thr Gly Arg Cys Leu
Arg Arg Glu Asn Leu Ala Arg 145 150 155 160 Gly Thr Cys Glu Ile Phe
Ala Trp Cys Pro Leu Glu Thr Ser Ser Arg 165 170 175 Pro Glu Glu Pro
Phe Leu Lys Glu Ala Glu Asp Phe Thr Ile Phe Ile 180 185 190 Lys Asn
His Ile Arg Phe Pro Lys Phe Asn Phe Ser Lys Ser Asn Val 195 200 205
Met Asp Val Lys Asp Arg Ser Phe Leu Lys Ser Cys His Phe Gly Pro 210
215 220 Lys Asn His Tyr Cys Pro Ile Phe Arg Leu Gly Ser Val Ile Arg
Trp 225 230 235 240 Ala Gly Ser Asp Phe Gln Asp Ile Ala Leu Glu Gly
Gly Val Ile Gly 245 250 255 Ile Asn Ile Glu Trp Asn Cys Asp Leu Asp
Lys Ala Ala Ser Glu Cys 260 265 270 His Pro His Tyr Ser Phe Ser Arg
Leu Asp Asn Lys Leu Ser Lys Ser 275 280 285 Val Ser Ser Gly Tyr Asn
Phe Arg Phe Ala Arg Tyr Tyr Arg Asp Ala 290 295 300 Ala Gly Val Glu
Phe Arg Thr Leu Met Lys Ala Tyr Gly Ile Arg Phe 305 310 315 320 Asp
Val Met Val Asn Gly Lys Gly Ala Phe Phe Cys Asp Leu Val Leu 325 330
335 Ile Tyr Leu Ile Lys Lys Arg Glu Phe Tyr Arg Asp Lys Lys Tyr Glu
340 345 350 Glu Val Arg Gly Leu Glu Asp Ser Ser Gln Glu Ala Glu Asp
Glu Ala 355 360 365 Ser Gly Leu Gly Leu Ser Glu Gln Leu Thr Ser Gly
Pro Gly Leu Leu 370 375 380 Gly Met Pro Glu Gln Gln Glu Leu Gln Glu
Pro Pro Glu Ala Lys Arg 385 390 395 400 Gly Ser Ser Ser Gln Lys Gly
Asn Gly Ser Val Cys Pro Gln Leu Leu 405 410 415 Glu Pro His Arg Ser
Thr 420 <210> SEQ ID NO 32 <211> LENGTH: 359
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CD72
<400> SEQUENCE: 32 Met Ala Glu Ala Ile Thr Tyr Ala Asp Leu
Arg Phe Val Lys Ala Pro 1 5 10 15 Leu Lys Lys Ser Ile Ser Ser Arg
Leu Gly Gln Asp Pro Gly Ala Asp 20 25 30 Asp Asp Gly Glu Ile Thr
Tyr Glu Asn Val Gln Val Pro Ala Val Leu 35 40 45 Gly Val Pro Ser
Ser Leu Ala Ser Ser Val Leu Gly Asp Lys Ala Ala 50 55 60 Val Lys
Ser Glu Gln Pro Thr Ala Ser Trp Arg Ala Val Thr Ser Pro 65 70 75 80
Ala Val Gly Arg Ile Leu Pro Cys Arg Thr Thr Cys Leu Arg Tyr Leu 85
90 95 Leu Leu Gly Leu Leu Leu Thr Cys Leu Leu Leu Gly Val Thr Ala
Ile 100 105 110 Cys Leu Gly Val Arg Tyr Leu Gln Val Ser Gln Gln Leu
Gln Gln Thr 115 120 125 Asn Arg Val Leu Glu Val Thr Asn Ser Ser Leu
Arg Gln Gln Leu Arg 130 135 140 Leu Lys Ile Thr Gln Leu Gly Gln Ser
Ala Glu Asp Leu Gln Gly Ser 145 150 155 160 Arg Arg Glu Leu Ala Gln
Ser Gln Glu Ala Leu Gln Val Glu Gln Arg 165 170 175 Ala His Gln Ala
Ala Glu Gly Gln Leu Gln Ala Cys Gln Ala Asp Arg 180 185 190 Gln Lys
Thr Lys Glu Thr Leu Gln Ser Glu Glu Gln Gln Arg Arg Ala 195 200 205
Leu Glu Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys Pro Phe Phe 210
215 220 Thr Cys Gly Ser Ala Asp Thr Cys Cys Pro Ser Gly Trp Ile Met
His 225 230 235 240 Gln Lys Ser Cys Phe Tyr Ile Ser Leu Thr Ser Lys
Asn Trp Gln Glu 245 250 255 Ser Gln Lys Gln Cys Glu Thr Leu Ser Ser
Lys Leu Ala Thr Phe Ser 260 265 270 Glu Ile Tyr Pro Gln Ser His Ser
Tyr Tyr Phe Leu Asn Ser Leu Leu 275 280 285 Pro Asn Gly Gly Ser Gly
Asn Ser Tyr Trp Thr Gly Leu Ser Ser Asn 290 295 300 Lys Asp Trp Lys
Leu Thr Asp Asp Thr Gln Arg Thr Arg Thr Tyr Ala 305 310 315 320 Gln
Ser Ser Lys Cys Asn Lys Val His Lys Thr Trp Ser Trp Trp Thr 325 330
335 Leu Glu Ser Glu Ser Cys Arg Ser Ser Leu Pro Tyr Ile Cys Glu Met
340 345 350 Thr Ala Phe Arg Phe Pro Asp 355 <210> SEQ ID NO
33 <211> LENGTH: 661 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: LY64 <400> SEQUENCE: 33 Met Ala Phe Asp Val Ser
Cys Phe Phe Trp Val Val Leu Phe Ser Ala 1 5 10 15 Gly Cys Lys Val
Ile Thr Ser Trp Asp Gln Met Cys Ile Glu Lys Glu 20 25 30 Ala Asn
Lys Thr Tyr Asn Cys Glu Asn Leu Gly Leu Ser Glu Ile Pro 35 40 45
Asp Thr Leu Pro Asn Thr Thr Glu Phe Leu Glu Phe Ser Phe Asn Phe 50
55 60 Leu Pro Thr Ile His Asn Arg Thr Phe Ser Arg Leu Met Asn Leu
Thr 65 70 75 80 Phe Leu Asp Leu Thr Arg Cys Gln Ile Asn Trp Ile His
Glu Asp Thr 85 90 95 Phe Gln Ser His His Gln Leu Ser Thr Leu Val
Leu Thr Gly Asn Pro 100 105 110 Leu Ile Phe Met Ala Glu Thr Ser Leu
Asn Gly Pro Lys Ser Leu Lys 115 120 125 His Leu Phe Leu Ile Gln Thr
Gly Ile Ser Asn Leu Glu Phe Ile Pro 130 135 140 Val His Asn Leu Glu
Asn Leu Glu Ser Leu Tyr Leu Gly Ser Asn His 145 150 155 160 Ile Ser
Ser Ile Lys Phe Pro Lys Asp Phe Pro Ala Arg Asn Leu Lys 165 170 175
Val Leu Asp Phe Gln Asn Asn Ala Ile His Tyr Ile Ser Arg Glu Asp 180
185 190 Met Arg Ser Leu Glu Gln Ala Ile Asn Leu Ser Leu Asn Phe Asn
Gly 195 200 205 Asn Asn Val Lys Gly Ile Glu Leu Gly Ala Phe Asp Ser
Thr Val Phe 210 215 220 Gln Ser Leu Asn Phe Gly Gly Thr Pro Asn Leu
Ser Val Ile Phe Asn 225 230 235 240 Gly Leu Gln Asn Ser Thr Thr Gln
Ser Leu Trp Leu Gly Thr Phe Glu 245 250 255 Asp Ile Asp Asp Glu Asp
Ile Ser Ser Ala Met Leu Lys Gly Leu Cys 260 265 270 Glu Met Ser Val
Glu Ser Leu Asn Leu Gln Glu His Arg Phe Ser Asp 275 280 285 Ile Ser
Ser Thr Thr Phe Gln Cys Phe Thr Gln Leu Gln Glu Leu Asp 290 295 300
Leu Thr Ala Thr His Leu Lys Gly Leu Pro Ser Gly Met Lys Gly Leu 305
310 315 320 Asn Leu Leu Lys Lys Leu Val Leu Ser Val Asn His Phe Asp
Gln Leu 325 330 335 Cys Gln Ile Ser Ala Ala Asn Phe Pro Ser Leu Thr
His Leu Tyr Ile 340 345 350 Arg Gly Asn Val Lys Lys Leu His Leu Gly
Val Gly Cys Leu Glu Lys 355 360 365 Leu Gly Asn Leu Gln Thr Leu Asp
Leu Ser His Asn Asp Ile Glu Ala 370 375 380 Ser Asp Cys Cys Ser Leu
Gln Leu Lys Asn Leu Ser His Leu Gln Thr 385 390 395 400 Leu Asn Leu
Ser His Asn Glu Pro Leu Gly Leu Gln Ser Gln Ala Phe 405 410 415 Lys
Glu Cys Pro Gln Leu Glu Leu Leu Asp Leu Ala Phe Thr Arg Leu 420 425
430 His Ile Asn Ala Pro Gln Ser Pro Phe Gln Asn Leu His Phe Leu Gln
435 440 445 Val Leu Asn Leu Thr Tyr Cys Phe Leu Asp Thr Ser Asn Gln
His Leu 450 455 460 Leu Ala Gly Leu Pro Val Leu Arg His Leu Asn Leu
Lys Gly Asn His 465 470 475 480 Phe Gln Asp Gly Thr Ile Thr Lys Thr
Asn Leu Leu Gln Thr Val Gly 485 490 495 Ser Leu Glu Val Leu Ile Leu
Ser Ser Cys Gly Leu Leu Ser Ile Asp 500 505 510 Gln Gln Ala Phe His
Ser Leu Gly Lys Met Ser His Val Asp Leu Ser 515 520 525 His Asn Ser
Leu Thr Cys Asp Ser Ile Asp Ser Leu Ser His Leu Lys 530 535 540 Gly
Ile Tyr Leu Asn Leu Ala Ala Asn Ser Ile Asn Ile Ile Ser Pro 545 550
555 560 Arg Leu Leu Pro Ile Leu Ser Gln Gln Ser Thr Ile Asn Leu Ser
His 565 570 575 Asn Pro Leu Asp Cys Thr Cys Ser Asn Ile His Phe Leu
Thr Trp Tyr 580 585 590 Lys Glu Asn Leu His Lys Leu Glu Gly Ser Glu
Glu Thr Thr Cys Ala 595 600 605 Asn Pro Pro Ser Leu Arg Gly Val Lys
Leu Ser Asp Val Lys Leu Ser 610 615 620 Cys Gly Ile Thr Ala Ile Gly
Ile Phe Phe Leu Ile Val Phe Leu Leu 625 630 635 640 Leu Leu Ala Ile
Leu Leu Phe Phe Ala Val Lys Tyr Leu Leu Arg Trp 645 650 655 Lys Tyr
Gln His Ile 660 <210> SEQ ID NO 34 <211> LENGTH: 429
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: FCRH1
<400> SEQUENCE: 34 Met Leu Pro Arg Leu Leu Leu Leu Ile Cys
Ala Pro Leu Cys Glu Pro 1 5 10 15 Ala Glu Leu Phe Leu Ile Ala Ser
Pro Ser His Pro Thr Glu Gly Ser 20 25 30 Pro Val Thr Leu Thr Cys
Lys Met Pro Phe Leu Gln Ser Ser Asp Ala 35 40 45 Gln Phe Gln Phe
Cys Phe Phe Arg Asp Thr Arg Ala Leu Gly Pro Gly 50 55 60 Trp Ser
Ser Ser Pro Lys Leu Gln Ile Ala Ala Met Trp Lys Glu Asp 65 70 75 80
Thr Gly Ser Tyr Trp Cys Glu Ala Gln Thr Met Ala Ser Lys Val Leu 85
90 95 Arg Ser Arg Arg Ser Gln Ile Asn Val His Arg Val Pro Val Ala
Asp 100 105 110 Val Ser Leu Glu Thr Gln Pro Pro Gly Gly Gln Val Met
Glu Gly Asp 115 120 125 Arg Leu Val Leu Ile Cys Ser Val Ala Met Gly
Thr Gly Asp Ile Thr 130 135 140 Phe Leu Trp Tyr Lys Gly Ala Val Gly
Leu Asn Leu Gln Ser Lys Thr 145 150 155 160 Gln Arg Ser Leu Thr Ala
Glu Tyr Glu Ile Pro Ser Val Arg Glu Ser 165 170 175 Asp Ala Glu Gln
Tyr Tyr Cys Val Ala Glu Asn Gly Tyr Gly Pro Ser 180 185 190 Pro Ser
Gly Leu Val Ser Ile Thr Val Arg Ile Pro Val Ser Arg Pro 195 200 205
Ile Leu Met Leu Arg Ala Pro Arg Ala Gln Ala Ala Val Glu Asp Val 210
215 220 Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Pro Pro Ile Leu
Tyr 225 230 235 240 Trp Phe Tyr His Glu Asp Ile Thr Leu Gly Ser Arg
Ser Ala Pro Ser 245 250 255 Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu
Thr Glu Glu His Ser Gly 260 265 270 Asn Tyr Ser Cys Glu Ala Asn Asn
Gly Leu Gly Ala Gln Arg Ser Glu 275 280 285 Ala Val Thr Leu Asn Phe
Thr Val Pro Thr Gly Ala Arg Ser Asn His 290 295 300 Leu Thr Ser Gly
Val Ile Glu Gly Leu Leu Ser Thr Leu Gly Pro Ala 305 310 315 320 Thr
Val Ala Leu Leu Phe Cys Tyr Gly Leu Lys Arg Lys Ile Gly Arg 325 330
335 Arg Ser Ala Arg Asp Pro Leu Arg Ser Leu Pro Ser Pro Leu Pro Gln
340 345 350 Glu Phe Thr Tyr Leu Asn Ser Pro Thr Pro Gly Gln Leu Gln
Pro Ile 355 360 365 Tyr Glu Asn Val Asn Val Val Ser Gly Asp Glu Val
Tyr Ser Leu Ala 370 375 380 Tyr Tyr Asn Gln Pro Glu Gln Glu Ser Val
Ala Ala Glu Thr Leu Gly 385 390 395 400 Thr His Met Glu Asp Lys Val
Ser Leu Asp Ile Tyr Ser Arg Leu Arg 405 410 415 Lys Ala Asn Ile Thr
Asp Val Asp Tyr Glu Asp Ala Met 420 425 <210> SEQ ID NO 35
<211> LENGTH: 977 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
IRTA2 <400> SEQUENCE: 35 Met Leu Leu Trp Val Ile Leu Leu Val
Leu Ala Pro Val Ser Gly Gln 1 5 10 15 Phe Ala Arg Thr Pro Arg Pro
Ile Ile Phe Leu Gln Pro Pro Trp Thr 20 25 30 Thr Val Phe Gln Gly
Glu Arg Val Thr Leu Thr Cys Lys Gly Phe Arg 35 40 45 Phe Tyr Ser
Pro Gln Lys Thr Lys Trp Tyr His Arg Tyr Leu Gly Lys 50 55 60 Glu
Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu Glu Val Gln Glu Ser 65 70
75 80 Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser Pro Leu Ser Ser Pro
Val 85 90 95 His Leu Asp Phe Ser Ser Ala Ser Leu Ile Leu Gln Ala
Pro Leu Ser 100 105 110 Val Phe Glu Gly Asp Ser Val Val Leu Arg Cys
Arg Ala Lys Ala Glu 115 120 125 Val Thr Leu Asn Asn Thr Ile Tyr Lys
Asn Asp Asn Val Leu Ala Phe 130 135 140 Leu Asn Lys Arg Thr Asp Phe
His Ile Pro His Ala Cys Leu Lys Asp 145 150 155 160 Asn Gly Ala Tyr
Arg Cys Thr Gly Tyr Lys Glu Ser Cys Cys Pro Val 165 170 175 Ser Ser
Asn Thr Val Lys Ile Gln Val Gln Glu Pro Phe Thr Arg Pro 180 185 190
Val Leu Arg Ala Ser Ser Phe Gln Pro Ile Ser Gly Asn Pro Val Thr 195
200 205 Leu Thr Cys Glu Thr Gln Leu Ser Leu Glu Arg Ser Asp Val Pro
Leu 210 215 220 Arg Phe Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu
Gly Trp Ser 225 230 235 240 Leu Ser Pro Asn Phe Gln Ile Thr Ala Met
Trp Ser Lys Asp Ser Gly 245 250 255 Phe Tyr Trp Cys Lys Ala Ala Thr
Met Pro His Ser Val Ile Ser Asp 260 265 270 Ser Pro Arg Ser Trp Ile
Gln Val Gln Ile Pro Ala Ser His Pro Val 275 280 285 Leu Thr Leu Ser
Pro Glu Lys Ala Leu Asn Phe Glu Gly Thr Lys Val 290 295 300 Thr Leu
His Cys Glu Thr Gln Glu Asp Ser Leu Arg Thr Leu Tyr Arg 305 310 315
320 Phe Tyr His Glu Gly Val Pro Leu Arg His Lys Ser Val Arg Cys Glu
325 330 335 Arg Gly Ala Ser Ile Ser Phe Ser Leu Thr Thr Glu Asn Ser
Gly Asn 340 345 350 Tyr Tyr Cys Thr Ala Asp Asn Gly Leu Gly Ala Lys
Pro Ser Lys Ala 355 360 365 Val Ser Leu Ser Val Thr Val Pro Val Ser
His Pro Val Leu Asn Leu 370 375 380 Ser Ser Pro Glu Asp Leu Ile Phe
Glu Gly Ala Lys Val Thr Leu His 385 390 395 400 Cys Glu Ala Gln Arg
Gly Ser Leu Pro Ile Leu Tyr Gln Phe His His 405 410 415 Glu Asp Ala
Ala Leu Glu Arg Arg Ser Ala Asn Ser Ala Gly Gly Val 420 425 430 Ala
Ile Ser Phe Ser Leu Thr Ala Glu His Ser Gly Asn Tyr Tyr Cys 435 440
445 Thr Ala Asp Asn Gly Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu
450 455 460 Ser Ile Thr Val Pro Val Ser His Pro Val Leu Thr Leu Ser
Ser Ala 465 470 475 480 Glu Ala Leu Thr Phe Glu Gly Ala Thr Val Thr
Leu His Cys Glu Val 485 490 495 Gln Arg Gly Ser Pro Gln Ile Leu Tyr
Gln Phe Tyr His Glu Asp Met 500 505 510 Pro Leu Trp Ser Ser Ser Thr
Pro Ser Val Gly Arg Val Ser Phe Ser 515 520 525 Phe Ser Leu Thr Glu
Gly His Ser Gly Asn Tyr Tyr Cys Thr Ala Asp 530 535 540 Asn Gly Phe
Gly Pro Gln Arg Ser Glu Val Val Ser Leu Phe Val Thr 545 550 555 560
Val Pro Val Ser Arg Pro Ile Leu Thr Leu Arg Val Pro Arg Ala Gln 565
570 575 Ala Val Val Gly Asp Leu Leu Glu Leu His Cys Glu Ala Pro Arg
Gly 580 585 590 Ser Pro Pro Ile Leu Tyr Trp Phe Tyr His Glu Asp Val
Thr Leu Gly 595 600 605 Ser Ser Ser Ala Pro Ser Gly Gly Glu Ala Ser
Phe Asn Leu Ser Leu 610 615 620 Thr Ala Glu His Ser Gly Asn Tyr Ser
Cys Glu Ala Asn Asn Gly Leu 625 630 635 640 Val Ala Gln His Ser Asp
Thr Ile Ser Leu Ser Val Ile Val Pro Val 645 650 655 Ser Arg Pro Ile
Leu Thr Phe Arg Ala Pro Arg Ala Gln Ala Val Val 660 665 670 Gly Asp
Leu Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Ser Pro 675 680 685
Ile Leu Tyr Trp Phe Tyr His Glu Asp Val Thr Leu Gly Lys Ile Ser 690
695 700 Ala Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr
Glu 705 710 715 720 His Ser Gly Ile Tyr Ser Cys Glu Ala Asp Asn Gly
Pro Glu Ala Gln 725 730 735 Arg Ser Glu Met Val Thr Leu Lys Val Ala
Val Pro Val Ser Arg Pro 740 745 750 Val Leu Thr Leu Arg Ala Pro Gly
Thr His Ala Ala Val Gly Asp Leu 755 760 765 Leu Glu Leu His Cys Glu
Ala Leu Arg Gly Ser Pro Leu Ile Leu Tyr 770 775 780 Arg Phe Phe His
Glu Asp Val Thr Leu Gly Asn Arg Ser Ser Pro Ser 785 790 795 800 Gly
Gly Ala Ser Leu Asn Leu Ser Leu Thr Ala Glu His Ser Gly Asn 805 810
815 Tyr Ser Cys Glu Ala Asp Asn Gly Leu Gly Ala Gln Arg Ser Glu Thr
820 825 830 Val Thr Leu Tyr Ile Thr Gly Leu Thr Ala Asn Arg Ser Gly
Pro Phe 835 840 845 Ala Thr Gly Val Ala Gly Gly Leu Leu Ser Ile Ala
Gly Leu Ala Ala 850 855 860 Gly Ala Leu Leu Leu Tyr Cys Trp Leu Ser
Arg Lys Ala Gly Arg Lys 865 870 875 880 Pro Ala Ser Asp Pro Ala Arg
Ser Pro Pro Asp Ser Asp Ser Gln Glu 885 890 895 Pro Thr Tyr His Asn
Val Pro Ala Trp Glu Glu Leu Gln Pro Val Tyr 900 905 910 Thr Asn Ala
Asn Pro Arg Gly Glu Asn Val Val Tyr Ser Glu Val Arg 915 920 925 Ile
Ile Gln Glu Lys Lys Lys His Ala Val Ala Ser Asp Pro Arg His 930 935
940 Leu Arg Asn Lys Gly Ser Pro Ile Ile Tyr Ser Glu Val Lys Val Ala
945 950 955 960 Ser Thr Pro Val Ser Gly Ser Leu Phe Leu Ala Ser Ser
Ala Pro His 965 970 975 Arg
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 35 <210>
SEQ ID NO 1 <211> LENGTH: 502 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: BMPR1B <400> SEQUENCE: 1 Met Leu Leu Arg
Ser Ala Gly Lys Leu Asn Val Gly Thr Lys Lys Glu 1 5 10 15 Asp Gly
Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys Val Leu Arg Cys 20 25 30
Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn Ile Cys Ser 35
40 45 Thr Asp Gly Tyr Cys Phe Thr Met Ile Glu Glu Asp Asp Ser Gly
Leu 50 55 60 Pro Val Val Thr Ser Gly Cys Leu Gly Leu Glu Gly Ser
Asp Phe Gln 65 70 75 80 Cys Arg Asp Thr Pro Ile Pro His Gln Arg Arg
Ser Ile Glu Cys Cys 85 90 95 Thr Glu Arg Asn Glu Cys Asn Lys Asp
Leu His Pro Thr Leu Pro Pro 100 105 110 Leu Lys Asn Arg Asp Phe Val
Asp Gly Pro Ile His His Arg Ala Leu 115 120 125 Leu Ile Ser Val Thr
Val Cys Ser Leu Leu Leu Val Leu Ile Ile Leu 130 135 140 Phe Cys Tyr
Phe Arg Tyr Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser 145 150 155 160
Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile Pro Pro Gly Glu Ser Leu 165
170 175 Arg Asp Leu Ile Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly
Leu 180 185 190 Pro Leu Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln
Met Val Lys 195 200 205 Gln Ile Gly Lys Gly Arg Tyr Gly Glu Val Trp
Met Gly Lys Trp Arg 210 215 220 Gly Glu Lys Val Ala Val Lys Val Phe
Phe Thr Thr Glu Glu Ala Ser 225 230 235 240 Trp Phe Arg Glu Thr Glu
Ile Tyr Gln Thr Val Leu Met Arg His Glu 245 250 255 Asn Ile Leu Gly
Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp 260 265 270 Thr Gln
Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr 275 280 285
Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala Lys Ser Met Leu Lys Leu 290
295 300 Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu His Thr Glu Ile
Phe 305 310 315 320 Ser Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp
Leu Lys Ser Lys 325 330 335 Asn Ile Leu Val Lys Lys Asn Gly Thr Cys
Cys Ile Ala Asp Leu Gly 340 345 350 Leu Ala Val Lys Phe Ile Ser Asp
Thr Asn Glu Val Asp Ile Pro Pro 355 360 365 Asn Thr Arg Val Gly Thr
Lys Arg Tyr Met Pro Pro Glu Val Leu Asp 370 375 380 Glu Ser Leu Asn
Arg Asn His Phe Gln Ser Tyr Ile Met Ala Asp Met 385 390 395 400 Tyr
Ser Phe Gly Leu Ile Leu Trp Glu Val Ala Arg Arg Cys Val Ser 405 410
415 Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu Val Pro
420 425 430 Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu Ile Val Cys Ile
Lys Lys 435 440 445 Leu Arg Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp
Glu Cys Leu Arg 450 455 460 Gln Met Gly Lys Leu Met Thr Glu Cys Trp
Ala His Asn Pro Ala Ser 465 470 475 480 Arg Leu Thr Ala Leu Arg Val
Lys Lys Thr Leu Ala Lys Met Ser Glu 485 490 495 Ser Gln Asp Ile Lys
Leu 500 <210> SEQ ID NO 2 <211> LENGTH: 507 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: SLC7A5 <400> SEQUENCE: 2 Met
Ala Gly Ala Gly Pro Lys Arg Arg Ala Leu Ala Ala Pro Ala Ala 1 5 10
15 Glu Glu Lys Glu Glu Ala Arg Glu Lys Met Leu Ala Ala Lys Ser Ala
20 25 30 Asp Gly Ser Ala Pro Ala Gly Glu Gly Glu Gly Val Thr Leu
Gln Arg 35 40 45 Asn Ile Thr Leu Leu Asn Gly Val Ala Ile Ile Val
Gly Thr Ile Ile 50 55 60 Gly Ser Gly Ile Phe Val Thr Pro Thr Gly
Val Leu Lys Glu Ala Gly 65 70 75 80 Ser Pro Gly Leu Ala Leu Val Val
Trp Ala Ala Cys Gly Val Phe Ser 85 90 95 Ile Val Gly Ala Leu Cys
Tyr Ala Glu Leu Gly Thr Thr Ile Ser Lys 100 105 110 Ser Gly Gly Asp
Tyr Ala Tyr Met Leu Glu Val Tyr Gly Ser Leu Pro 115 120 125 Ala Phe
Leu Lys Leu Trp Ile Glu Leu Leu Ile Ile Arg Pro Ser Ser 130 135 140
Gln Tyr Ile Val Ala Leu Val Phe Ala Thr Tyr Leu Leu Lys Pro Leu 145
150 155 160 Phe Pro Thr Cys Pro Val Pro Glu Glu Ala Ala Lys Leu Val
Ala Cys 165 170 175 Leu Cys Val Leu Leu Leu Thr Ala Val Asn Cys Tyr
Ser Val Lys Ala 180 185 190 Ala Thr Arg Val Gln Asp Ala Phe Ala Ala
Ala Lys Leu Leu Ala Leu 195 200 205 Ala Leu Ile Ile Leu Leu Gly Phe
Val Gln Ile Gly Lys Gly Val Val 210 215 220 Ser Asn Leu Asp Pro Asn
Phe Ser Phe Glu Gly Thr Lys Leu Asp Val 225 230 235 240 Gly Asn Ile
Val Leu Ala Leu Tyr Ser Gly Leu Phe Ala Tyr Gly Gly 245 250 255 Trp
Asn Tyr Leu Asn Phe Val Thr Glu Glu Met Ile Asn Pro Tyr Arg 260 265
270 Asn Leu Pro Leu Ala Ile Ile Ile Ser Leu Pro Ile Val Thr Leu Val
275 280 285 Tyr Val Leu Thr Asn Leu Ala Tyr Phe Thr Thr Leu Ser Thr
Glu Gln 290 295 300 Met Leu Ser Ser Glu Ala Val Ala Val Asp Phe Gly
Asn Tyr His Leu 305 310 315 320 Gly Val Met Ser Trp Ile Ile Pro Val
Phe Val Gly Leu Ser Cys Phe 325 330 335 Gly Ser Val Asn Gly Ser Leu
Phe Thr Ser Ser Arg Leu Phe Phe Val 340 345 350 Gly Ser Arg Glu Gly
His Leu Pro Ser Ile Leu Ser Met Ile His Pro 355 360 365 Gln Leu Leu
Thr Pro Val Pro Ser Leu Val Phe Thr Cys Val Met Thr 370 375 380 Leu
Leu Tyr Ala Phe Ser Lys Asp Ile Phe Ser Val Ile Asn Phe Phe 385 390
395 400 Ser Phe Phe Asn Trp Leu Cys Val Ala Leu Ala Ile Ile Gly Met
Ile 405 410 415 Trp Leu Arg His Arg Lys Pro Glu Leu Glu Arg Pro Ile
Lys Val Asn 420 425 430 Leu Ala Leu Pro Val Phe Phe Ile Leu Ala Cys
Leu Phe Leu Ile Ala 435 440 445 Val Ser Phe Trp Lys Thr Pro Val Glu
Cys Gly Ile Gly Phe Thr Ile 450 455 460 Ile Leu Ser Gly Leu Pro Val
Tyr Phe Phe Gly Val Trp Trp Lys Asn 465 470 475 480 Lys Pro Lys Trp
Leu Leu Gln Gly Ile Phe Ser Thr Thr Val Leu Cys 485 490 495 Gln Lys
Leu Met Gln Val Val Pro Gln Glu Thr 500 505 <210> SEQ ID NO 3
<211> LENGTH: 339 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
STEAP1 <400> SEQUENCE: 3 Met Glu Ser Arg Lys Asp Ile Thr Asn
Gln Glu Glu Leu Trp Lys Met 1 5 10 15 Lys Pro Arg Arg Asn Leu Glu
Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30 Gly Glu Thr Ser Met
Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40 45 Thr Ala His
Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr 50 55 60 Gln
Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile 65 70
75 80 Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile
His 85 90 95 Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile
Pro Ile Leu 100 105 110 Val Ile Asn Lys Val Leu Pro Met Val Ser Ile
Thr Leu Leu Ala Leu 115 120 125
Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130
135 140 Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu
Thr 145 150 155 160 Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala
Val Leu His Ala 165 170 175 Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg
Ser Tyr Arg Tyr Lys Leu 180 185 190 Leu Asn Trp Ala Tyr Gln Gln Val
Gln Gln Asn Lys Glu Asp Ala Trp 195 200 205 Ile Glu His Asp Val Trp
Arg Met Glu Ile Tyr Val Ser Leu Gly Ile 210 215 220 Val Gly Leu Ala
Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser 225 230 235 240 Val
Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys 245 250
255 Leu Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His Ala Leu Ile Phe
260 265 270 Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr
Thr Pro 275 280 285 Pro Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val
Val Leu Ile Phe 290 295 300 Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg
Lys Lys Ile Leu Lys Ile 305 310 315 320 Arg His Gly Trp Glu Asp Val
Thr Lys Ile Asn Lys Thr Glu Ile Cys 325 330 335 Ser Gln Leu
<210> SEQ ID NO 4 <211> LENGTH: 6995 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: MUC16 <400> SEQUENCE: 4 Pro
Val Thr Ser Leu Leu Thr Pro Gly Leu Val Ile Thr Thr Asp Arg 1 5 10
15 Met Gly Ile Ser Arg Glu Pro Gly Thr Ser Ser Thr Ser Asn Leu Ser
20 25 30 Ser Thr Ser His Glu Arg Leu Thr Thr Leu Glu Asp Thr Val
Asp Thr 35 40 45 Glu Ala Met Gln Pro Ser Thr His Thr Ala Val Thr
Asn Val Arg Thr 50 55 60 Ser Ile Ser Gly His Glu Ser Gln Ser Ser
Val Leu Ser Asp Ser Glu 65 70 75 80 Thr Pro Lys Ala Thr Ser Pro Met
Gly Thr Thr Tyr Thr Met Gly Glu 85 90 95 Thr Ser Val Ser Ile Ser
Thr Ser Asp Phe Phe Glu Thr Ser Arg Ile 100 105 110 Gln Ile Glu Pro
Thr Ser Ser Leu Thr Ser Gly Leu Arg Glu Thr Ser 115 120 125 Ser Ser
Glu Arg Ile Ser Ser Ala Thr Glu Gly Ser Thr Val Leu Ser 130 135 140
Glu Val Pro Ser Gly Ala Thr Thr Glu Val Ser Arg Thr Glu Val Ile 145
150 155 160 Ser Ser Arg Gly Thr Ser Met Ser Gly Pro Asp Gln Phe Thr
Ile Ser 165 170 175 Pro Asp Ile Ser Thr Glu Ala Ile Thr Arg Leu Ser
Thr Ser Pro Ile 180 185 190 Met Thr Glu Ser Ala Glu Ser Ala Ile Thr
Ile Glu Thr Gly Ser Pro 195 200 205 Gly Ala Thr Ser Glu Gly Thr Leu
Thr Leu Asp Thr Ser Thr Thr Thr 210 215 220 Phe Trp Ser Gly Thr His
Ser Thr Ala Ser Pro Gly Phe Ser His Ser 225 230 235 240 Glu Met Thr
Thr Leu Met Ser Arg Thr Pro Gly Asp Val Pro Trp Pro 245 250 255 Ser
Leu Pro Ser Val Glu Glu Ala Ser Ser Val Ser Ser Ser Leu Ser 260 265
270 Ser Pro Ala Met Thr Ser Thr Ser Phe Phe Ser Thr Leu Pro Glu Ser
275 280 285 Ile Ser Ser Ser Pro His Pro Val Thr Ala Leu Leu Thr Leu
Gly Pro 290 295 300 Val Lys Thr Thr Asp Met Leu Arg Thr Ser Ser Glu
Pro Glu Thr Ser 305 310 315 320 Ser Pro Pro Asn Leu Ser Ser Thr Ser
Ala Glu Ile Leu Ala Thr Ser 325 330 335 Glu Val Thr Lys Asp Arg Glu
Lys Ile His Pro Ser Ser Asn Thr Pro 340 345 350 Val Val Asn Val Gly
Thr Val Ile Tyr Lys His Leu Ser Pro Ser Ser 355 360 365 Val Leu Ala
Asp Leu Val Thr Thr Lys Pro Thr Ser Pro Met Ala Thr 370 375 380 Thr
Ser Thr Leu Gly Asn Thr Ser Val Ser Thr Ser Thr Pro Ala Phe 385 390
395 400 Pro Glu Thr Met Met Thr Gln Pro Thr Ser Ser Leu Thr Ser Gly
Leu 405 410 415 Arg Glu Ile Ser Thr Ser Gln Glu Thr Ser Ser Ala Thr
Glu Arg Ser 420 425 430 Ala Ser Leu Ser Gly Met Pro Thr Gly Ala Thr
Thr Lys Val Ser Arg 435 440 445 Thr Glu Ala Leu Ser Leu Gly Arg Thr
Ser Thr Pro Gly Pro Ala Gln 450 455 460 Ser Thr Ile Ser Pro Glu Ile
Ser Thr Glu Thr Ile Thr Arg Ile Ser 465 470 475 480 Thr Pro Leu Thr
Thr Thr Gly Ser Ala Glu Met Thr Ile Thr Pro Lys 485 490 495 Thr Gly
His Ser Gly Ala Ser Ser Gln Gly Thr Phe Thr Leu Asp Thr 500 505 510
Ser Ser Arg Ala Ser Trp Pro Gly Thr His Ser Ala Ala Thr His Arg 515
520 525 Ser Pro His Ser Gly Met Thr Thr Pro Met Ser Arg Gly Pro Glu
Asp 530 535 540 Val Ser Trp Pro Ser Arg Pro Ser Val Glu Lys Thr Ser
Pro Pro Ser 545 550 555 560 Ser Leu Val Ser Leu Ser Ala Val Thr Ser
Pro Ser Pro Leu Tyr Ser 565 570 575 Thr Pro Ser Glu Ser Ser His Ser
Ser Pro Leu Arg Val Thr Ser Leu 580 585 590 Phe Thr Pro Val Met Met
Lys Thr Thr Asp Met Leu Asp Thr Ser Leu 595 600 605 Glu Pro Val Thr
Thr Ser Pro Pro Ser Met Asn Ile Thr Ser Asp Glu 610 615 620 Ser Leu
Ala Thr Ser Lys Ala Thr Met Glu Thr Glu Ala Ile Gln Leu 625 630 635
640 Ser Glu Asn Thr Ala Val Thr Gln Met Gly Thr Ile Ser Ala Arg Gln
645 650 655 Glu Phe Tyr Ser Ser Tyr Pro Gly Leu Pro Glu Pro Ser Lys
Val Thr 660 665 670 Ser Pro Val Val Thr Ser Ser Thr Ile Lys Asp Ile
Val Ser Thr Thr 675 680 685 Ile Pro Ala Ser Ser Glu Ile Thr Arg Ile
Glu Met Glu Ser Thr Ser 690 695 700 Thr Leu Thr Pro Thr Pro Arg Glu
Thr Ser Thr Ser Gln Glu Ile His 705 710 715 720 Ser Ala Thr Lys Pro
Ser Thr Val Pro Tyr Lys Ala Leu Thr Ser Ala 725 730 735 Thr Ile Glu
Asp Ser Met Thr Gln Val Met Ser Ser Ser Arg Gly Pro 740 745 750 Ser
Pro Asp Gln Ser Thr Met Ser Gln Asp Ile Ser Thr Glu Val Ile 755 760
765 Thr Arg Leu Ser Thr Ser Pro Ile Lys Thr Glu Ser Thr Glu Met Thr
770 775 780 Ile Thr Thr Gln Thr Gly Ser Pro Gly Ala Thr Ser Arg Gly
Thr Leu 785 790 795 800 Thr Leu Asp Thr Ser Thr Thr Phe Met Ser Gly
Thr His Ser Thr Ala 805 810 815 Ser Gln Gly Phe Ser His Ser Gln Met
Thr Ala Leu Met Ser Arg Thr 820 825 830 Pro Gly Glu Val Pro Trp Leu
Ser His Pro Ser Val Glu Glu Ala Ser 835 840 845 Ser Ala Ser Phe Ser
Leu Ser Ser Pro Val Met Thr Ser Ser Ser Pro 850 855 860 Val Ser Ser
Thr Leu Pro Asp Ser Ile His Ser Ser Ser Leu Pro Val 865 870 875 880
Thr Ser Leu Leu Thr Ser Gly Leu Val Lys Thr Thr Glu Leu Leu Gly 885
890 895 Thr Ser Ser Glu Pro Glu Thr Ser Ser Pro Pro Asn Leu Ser Ser
Thr 900 905 910 Ser Ala Glu Ile Leu Ala Thr Thr Glu Val Thr Thr Asp
Thr Glu Lys 915 920 925 Leu Glu Met Thr Asn Val Val Thr Ser Gly Tyr
Thr His Glu Ser Pro 930 935 940 Ser Ser Val Leu Ala Asp Ser Val Thr
Thr Lys Ala Thr Ser Ser Met 945 950 955 960 Gly Ile Thr Tyr Pro Thr
Gly Asp Thr Asn Val Leu Thr Ser Thr Pro 965 970 975 Ala Phe Ser Asp
Thr Ser Arg Ile Gln Thr Lys Ser Lys Leu Ser Leu 980 985 990 Thr Pro
Gly Leu Met Glu Thr Ser Ile Ser Glu Glu Thr Ser Ser Ala 995 1000
1005 Thr Glu Lys Ser Thr Val Leu Ser Ser Val Pro Thr Gly Ala Thr
Thr 1010 1015 1020 Glu Val Ser Arg Thr Glu Ala Ile Ser Ser Ser Arg
Thr Ser Ile Pro 1025 1030 1035 1040 Gly Pro Ala Gln Ser Thr Met Ser
Ser Asp Thr Ser Met Glu Thr Ile 1045 1050 1055 Thr Arg Ile Ser Thr
Pro Leu Thr Arg Lys Glu Ser Thr Asp Met Ala
1060 1065 1070 Ile Thr Pro Lys Thr Gly Pro Ser Gly Ala Thr Ser Gln
Gly Thr Phe 1075 1080 1085 Thr Leu Asp Ser Ser Ser Thr Ala Ser Trp
Pro Gly Thr His Ser Ala 1090 1095 1100 Thr Thr Gln Arg Phe Pro Arg
Ser Val Val Thr Thr Pro Met Ser Arg 1105 1110 1115 1120 Gly Pro Glu
Asp Val Ser Trp Pro Ser Pro Leu Ser Val Glu Lys Asn 1125 1130 1135
Ser Pro Pro Ser Ser Leu Val Ser Ser Ser Ser Val Thr Ser Pro Ser
1140 1145 1150 Pro Leu Tyr Ser Thr Pro Ser Gly Ser Ser His Ser Ser
Pro Val Pro 1155 1160 1165 Val Thr Ser Leu Phe Thr Ser Ile Met Met
Lys Ala Thr Asp Met Leu 1170 1175 1180 Asp Ala Ser Leu Glu Pro Glu
Thr Thr Ser Ala Pro Asn Met Asn Ile 1185 1190 1195 1200 Thr Ser Asp
Glu Ser Leu Ala Ala Ser Lys Ala Thr Thr Glu Thr Glu 1205 1210 1215
Ala Ile His Val Phe Glu Asn Thr Ala Ala Ser His Val Glu Thr Thr
1220 1225 1230 Ser Ala Thr Glu Glu Leu Tyr Ser Ser Ser Pro Gly Phe
Ser Glu Pro 1235 1240 1245 Thr Lys Val Ile Ser Pro Val Val Thr Ser
Ser Ser Ile Arg Asp Asn 1250 1255 1260 Met Val Ser Thr Thr Met Pro
Gly Ser Ser Gly Ile Thr Arg Ile Glu 1265 1270 1275 1280 Ile Glu Ser
Met Ser Ser Leu Thr Pro Gly Leu Arg Glu Thr Arg Thr 1285 1290 1295
Ser Gln Asp Ile Thr Ser Ser Thr Glu Thr Ser Thr Val Leu Tyr Lys
1300 1305 1310 Met Pro Ser Gly Ala Thr Pro Glu Val Ser Arg Thr Glu
Val Met Pro 1315 1320 1325 Ser Ser Arg Thr Ser Ile Pro Gly Pro Ala
Gln Ser Thr Met Ser Leu 1330 1335 1340 Asp Ile Ser Asp Glu Val Val
Thr Arg Leu Ser Thr Ser Pro Ile Met 1345 1350 1355 1360 Thr Glu Ser
Ala Glu Ile Thr Ile Thr Thr Gln Thr Gly Tyr Ser Leu 1365 1370 1375
Ala Thr Ser Gln Val Thr Leu Pro Leu Gly Thr Ser Met Thr Phe Leu
1380 1385 1390 Ser Gly Thr His Ser Thr Met Ser Gln Gly Leu Ser His
Ser Glu Met 1395 1400 1405 Thr Asn Leu Met Ser Arg Gly Pro Glu Ser
Leu Ser Trp Thr Ser Pro 1410 1415 1420 Arg Phe Val Glu Thr Thr Arg
Ser Ser Ser Ser Leu Thr Ser Leu Pro 1425 1430 1435 1440 Leu Thr Thr
Ser Leu Ser Pro Val Ser Ser Thr Leu Leu Asp Ser Ser 1445 1450 1455
Pro Ser Ser Pro Leu Pro Val Thr Ser Leu Ile Leu Pro Gly Leu Val
1460 1465 1470 Lys Thr Thr Glu Val Leu Asp Thr Ser Ser Glu Pro Lys
Thr Ser Ser 1475 1480 1485 Ser Pro Asn Leu Ser Ser Thr Ser Val Glu
Ile Pro Ala Thr Ser Glu 1490 1495 1500 Ile Met Thr Asp Thr Glu Lys
Ile His Pro Ser Ser Asn Thr Ala Val 1505 1510 1515 1520 Ala Lys Val
Arg Thr Ser Ser Ser Val His Glu Ser His Ser Ser Val 1525 1530 1535
Leu Ala Asp Ser Glu Thr Thr Ile Thr Ile Pro Ser Met Gly Ile Thr
1540 1545 1550 Ser Ala Val Glu Asp Thr Thr Val Phe Thr Ser Asn Pro
Ala Phe Ser 1555 1560 1565 Glu Thr Arg Arg Ile Pro Thr Glu Pro Thr
Phe Ser Leu Thr Pro Gly 1570 1575 1580 Phe Arg Glu Thr Ser Thr Ser
Glu Glu Thr Thr Ser Ile Thr Glu Thr 1585 1590 1595 1600 Ser Ala Val
Leu Phe Gly Val Pro Thr Ser Ala Thr Thr Glu Val Ser 1605 1610 1615
Met Thr Glu Ile Met Ser Ser Asn Arg Thr His Ile Pro Asp Ser Asp
1620 1625 1630 Gln Ser Thr Met Ser Pro Asp Ile Ile Thr Glu Val Ile
Thr Arg Leu 1635 1640 1645 Ser Ser Ser Ser Met Met Ser Glu Ser Thr
Gln Met Thr Ile Thr Thr 1650 1655 1660 Gln Lys Ser Ser Pro Gly Ala
Thr Ala Gln Ser Thr Leu Thr Leu Ala 1665 1670 1675 1680 Thr Thr Thr
Ala Pro Leu Ala Arg Thr His Ser Thr Val Pro Pro Arg 1685 1690 1695
Phe Leu His Ser Glu Met Thr Thr Leu Met Ser Arg Ser Pro Glu Asn
1700 1705 1710 Pro Ser Trp Lys Ser Ser Pro Phe Val Glu Lys Thr Ser
Ser Ser Ser 1715 1720 1725 Ser Leu Leu Ser Leu Pro Val Thr Thr Ser
Pro Ser Val Ser Ser Thr 1730 1735 1740 Leu Pro Gln Ser Ile Pro Ser
Ser Ser Phe Ser Val Thr Ser Leu Leu 1745 1750 1755 1760 Thr Pro Gly
Met Val Lys Thr Thr Asp Thr Ser Thr Glu Pro Gly Thr 1765 1770 1775
Ser Leu Ser Pro Asn Leu Ser Gly Thr Ser Val Glu Ile Leu Ala Ala
1780 1785 1790 Ser Glu Val Thr Thr Asp Thr Glu Lys Ile His Pro Ser
Ser Ser Met 1795 1800 1805 Ala Val Thr Asn Val Gly Thr Thr Ser Ser
Gly His Glu Leu Tyr Ser 1810 1815 1820 Ser Val Ser Ile His Ser Glu
Pro Ser Lys Ala Thr Tyr Pro Val Gly 1825 1830 1835 1840 Thr Pro Ser
Ser Met Ala Glu Thr Ser Ile Ser Thr Ser Met Pro Ala 1845 1850 1855
Asn Phe Glu Thr Thr Gly Phe Glu Ala Glu Pro Phe Ser His Leu Thr
1860 1865 1870 Ser Gly Leu Arg Lys Thr Asn Met Ser Leu Asp Thr Ser
Ser Val Thr 1875 1880 1885 Pro Thr Asn Thr Pro Ser Ser Pro Gly Ser
Thr His Leu Leu Gln Ser 1890 1895 1900 Ser Lys Thr Asp Phe Thr Ser
Ser Ala Lys Thr Ser Ser Pro Asp Trp 1905 1910 1915 1920 Pro Pro Ala
Ser Gln Tyr Thr Glu Ile Pro Val Asp Ile Ile Thr Pro 1925 1930 1935
Phe Asn Ala Ser Pro Ser Ile Thr Glu Ser Thr Gly Ile Thr Ser Phe
1940 1945 1950 Pro Glu Ser Arg Phe Thr Met Ser Val Thr Glu Ser Thr
His His Leu 1955 1960 1965 Ser Thr Asp Leu Leu Pro Ser Ala Glu Thr
Ile Ser Thr Gly Thr Val 1970 1975 1980 Met Pro Ser Leu Ser Glu Ala
Met Thr Ser Phe Ala Thr Thr Gly Val 1985 1990 1995 2000 Pro Arg Ala
Ile Ser Gly Ser Gly Ser Pro Phe Ser Arg Thr Glu Ser 2005 2010 2015
Gly Pro Gly Asp Ala Thr Leu Ser Thr Ile Ala Glu Ser Leu Pro Ser
2020 2025 2030 Ser Thr Pro Val Pro Phe Ser Ser Ser Thr Phe Thr Thr
Thr Asp Ser 2035 2040 2045 Ser Thr Ile Pro Ala Leu His Glu Ile Thr
Ser Ser Ser Ala Thr Pro 2050 2055 2060 Tyr Arg Val Asp Thr Ser Leu
Gly Thr Glu Ser Ser Thr Thr Glu Gly 2065 2070 2075 2080 Arg Leu Val
Met Val Ser Thr Leu Asp Thr Ser Ser Gln Pro Gly Arg 2085 2090 2095
Thr Ser Ser Ser Pro Ile Leu Asp Thr Arg Met Thr Glu Ser Val Glu
2100 2105 2110 Leu Gly Thr Val Thr Ser Ala Tyr Gln Val Pro Ser Leu
Ser Thr Arg 2115 2120 2125 Leu Thr Arg Thr Asp Gly Ile Met Glu His
Ile Thr Lys Ile Pro Asn 2130 2135 2140 Glu Ala Ala His Arg Gly Thr
Ile Arg Pro Val Lys Gly Pro Gln Thr 2145 2150 2155 2160 Ser Thr Ser
Pro Ala Ser Pro Lys Gly Leu His Thr Gly Gly Thr Lys 2165 2170 2175
Arg Met Glu Thr Thr Thr Thr Ala Leu Lys Thr Thr Thr Thr Ala Leu
2180 2185 2190 Lys Thr Thr Ser Arg Ala Thr Leu Thr Thr Ser Val Tyr
Thr Pro Thr 2195 2200 2205 Leu Gly Thr Leu Thr Pro Leu Asn Ala Ser
Met Gln Met Ala Ser Thr 2210 2215 2220 Ile Pro Thr Glu Met Met Ile
Thr Thr Pro Tyr Val Phe Pro Asp Val 2225 2230 2235 2240 Pro Glu Thr
Thr Ser Ser Leu Ala Thr Ser Leu Gly Ala Glu Thr Ser 2245 2250 2255
Thr Ala Leu Pro Arg Thr Thr Pro Ser Val Phe Asn Arg Glu Ser Glu
2260 2265 2270 Thr Thr Ala Ser Leu Val Ser Arg Ser Gly Ala Glu Arg
Ser Pro Val 2275 2280 2285 Ile Gln Thr Leu Asp Val Ser Ser Ser Glu
Pro Asp Thr Thr Ala Ser 2290 2295 2300 Trp Val Ile His Pro Ala Glu
Thr Ile Pro Thr Val Ser Lys Thr Thr 2305 2310 2315 2320 Pro Asn Phe
Phe His Ser Glu Leu Asp Thr Val Ser Ser Thr Ala Thr 2325 2330 2335
Ser His Gly Ala Asp Val Ser Ser Ala Ile Pro Thr Asn Ile Ser Pro
2340 2345 2350 Ser Glu Leu Asp Ala Leu Thr Pro Leu Val Thr Ile Ser
Gly Thr Asp 2355 2360 2365 Thr Ser Thr Thr Phe Pro Thr Leu Thr Lys
Ser Pro His Glu Thr Glu 2370 2375 2380 Thr Arg Thr Thr Trp Leu Thr
His Pro Ala Glu Thr Ser Ser Thr Ile 2385 2390 2395 2400
Pro Arg Thr Ile Pro Asn Phe Ser His His Glu Ser Asp Ala Thr Pro
2405 2410 2415 Ser Ile Ala Thr Ser Pro Gly Ala Glu Thr Ser Ser Ala
Ile Pro Ile 2420 2425 2430 Met Thr Val Ser Pro Gly Ala Glu Asp Leu
Val Thr Ser Gln Val Thr 2435 2440 2445 Ser Ser Gly Thr Asp Arg Asn
Met Thr Ile Pro Thr Leu Thr Leu Ser 2450 2455 2460 Pro Gly Glu Pro
Lys Thr Ile Ala Ser Leu Val Thr His Pro Glu Ala 2465 2470 2475 2480
Gln Thr Ser Ser Ala Ile Pro Thr Ser Thr Ile Ser Pro Ala Val Ser
2485 2490 2495 Arg Leu Val Thr Ser Met Val Thr Ser Leu Ala Ala Lys
Thr Ser Thr 2500 2505 2510 Thr Asn Arg Ala Leu Thr Asn Ser Pro Gly
Glu Pro Ala Thr Thr Val 2515 2520 2525 Ser Leu Val Thr His Ser Ala
Gln Thr Ser Pro Thr Val Pro Trp Thr 2530 2535 2540 Thr Ser Ile Phe
Phe His Ser Lys Ser Asp Thr Thr Pro Ser Met Thr 2545 2550 2555 2560
Thr Ser His Gly Ala Glu Ser Ser Ser Ala Val Pro Thr Pro Thr Val
2565 2570 2575 Ser Thr Glu Val Pro Gly Val Val Thr Pro Leu Val Thr
Ser Ser Arg 2580 2585 2590 Ala Val Ile Ser Thr Thr Ile Pro Ile Leu
Thr Leu Ser Pro Gly Glu 2595 2600 2605 Pro Glu Thr Thr Pro Ser Met
Ala Thr Ser His Gly Glu Glu Ala Ser 2610 2615 2620 Ser Ala Ile Pro
Thr Pro Thr Val Ser Pro Gly Val Pro Gly Val Val 2625 2630 2635 2640
Thr Ser Leu Val Thr Ser Ser Arg Ala Val Thr Ser Thr Thr Ile Pro
2645 2650 2655 Ile Leu Thr Phe Ser Leu Gly Glu Pro Glu Thr Thr Pro
Ser Met Ala 2660 2665 2670 Thr Ser His Gly Thr Glu Ala Gly Ser Ala
Val Pro Thr Val Leu Pro 2675 2680 2685 Glu Val Pro Gly Met Val Thr
Ser Leu Val Ala Ser Ser Arg Ala Val 2690 2695 2700 Thr Ser Thr Thr
Leu Pro Thr Leu Thr Leu Ser Pro Gly Glu Pro Glu 2705 2710 2715 2720
Thr Thr Pro Ser Met Ala Thr Ser His Gly Ala Glu Ala Ser Ser Thr
2725 2730 2735 Val Pro Thr Val Ser Pro Glu Val Pro Gly Val Val Thr
Ser Leu Val 2740 2745 2750 Thr Ser Ser Ser Gly Val Asn Ser Thr Ser
Ile Pro Thr Leu Ile Leu 2755 2760 2765 Ser Pro Gly Glu Leu Glu Thr
Thr Pro Ser Met Ala Thr Ser His Gly 2770 2775 2780 Ala Glu Ala Ser
Ser Ala Val Pro Thr Pro Thr Val Ser Pro Gly Val 2785 2790 2795 2800
Ser Gly Val Val Thr Pro Leu Val Thr Ser Ser Arg Ala Val Thr Ser
2805 2810 2815 Thr Thr Ile Pro Ile Leu Thr Leu Ser Ser Ser Glu Pro
Glu Thr Thr 2820 2825 2830 Pro Ser Met Ala Thr Ser His Gly Val Glu
Ala Ser Ser Ala Val Leu 2835 2840 2845 Thr Val Ser Pro Glu Val Pro
Gly Met Val Thr Phe Leu Val Thr Ser 2850 2855 2860 Ser Arg Ala Val
Thr Ser Thr Thr Ile Pro Thr Leu Thr Ile Ser Ser 2865 2870 2875 2880
Asp Glu Pro Glu Thr Thr Thr Ser Leu Val Thr His Ser Glu Ala Lys
2885 2890 2895 Met Ile Ser Ala Ile Pro Thr Leu Gly Val Ser Pro Thr
Val Gln Gly 2900 2905 2910 Leu Val Thr Ser Leu Val Thr Ser Ser Gly
Ser Glu Thr Ser Ala Phe 2915 2920 2925 Ser Asn Leu Thr Val Ala Ser
Ser Gln Pro Glu Thr Ile Asp Ser Trp 2930 2935 2940 Val Ala His Pro
Gly Thr Glu Ala Ser Ser Val Val Pro Thr Leu Thr 2945 2950 2955 2960
Val Ser Thr Gly Glu Pro Phe Thr Asn Ile Ser Leu Val Thr His Pro
2965 2970 2975 Ala Glu Ser Ser Ser Thr Leu Pro Arg Thr Thr Ser Arg
Phe Ser His 2980 2985 2990 Ser Glu Leu Asp Thr Met Pro Ser Thr Val
Thr Ser Pro Glu Ala Glu 2995 3000 3005 Ser Ser Ser Ala Ile Ser Thr
Thr Ile Ser Pro Gly Ile Pro Gly Val 3010 3015 3020 Leu Thr Ser Leu
Val Thr Ser Ser Gly Arg Asp Ile Ser Ala Thr Phe 3025 3030 3035 3040
Pro Thr Val Pro Glu Ser Pro His Glu Ser Glu Ala Thr Ala Ser Trp
3045 3050 3055 Val Thr His Pro Ala Val Thr Ser Thr Thr Val Pro Arg
Thr Thr Pro 3060 3065 3070 Asn Tyr Ser His Ser Glu Pro Asp Thr Thr
Pro Ser Ile Ala Thr Ser 3075 3080 3085 Pro Gly Ala Glu Ala Thr Ser
Asp Phe Pro Thr Ile Thr Val Ser Pro 3090 3095 3100 Asp Val Pro Asp
Met Val Thr Ser Gln Val Thr Ser Ser Gly Thr Asp 3105 3110 3115 3120
Thr Ser Ile Thr Ile Pro Thr Leu Thr Leu Ser Ser Gly Glu Pro Glu
3125 3130 3135 Thr Thr Thr Ser Phe Ile Thr Tyr Ser Glu Thr His Thr
Ser Ser Ala 3140 3145 3150 Ile Pro Thr Leu Pro Val Ser Pro Asp Ala
Ser Lys Met Leu Thr Ser 3155 3160 3165 Leu Val Ile Ser Ser Gly Thr
Asp Ser Thr Thr Thr Phe Pro Thr Leu 3170 3175 3180 Thr Glu Thr Pro
Tyr Glu Pro Glu Thr Thr Ala Ile Gln Leu Ile His 3185 3190 3195 3200
Pro Ala Glu Thr Asn Thr Met Val Pro Arg Thr Thr Pro Lys Phe Ser
3205 3210 3215 His Ser Lys Ser Asp Thr Thr Leu Pro Val Ala Ile Thr
Ser Pro Gly 3220 3225 3230 Pro Glu Ala Ser Ser Ala Val Ser Thr Thr
Thr Ile Ser Pro Asp Met 3235 3240 3245 Ser Asp Leu Val Thr Ser Leu
Val Pro Ser Ser Gly Thr Asp Thr Ser 3250 3255 3260 Thr Thr Phe Pro
Thr Leu Ser Glu Thr Pro Tyr Glu Pro Glu Thr Thr 3265 3270 3275 3280
Ala Thr Trp Leu Thr His Pro Ala Glu Thr Ser Thr Thr Val Ser Gly
3285 3290 3295 Thr Ile Pro Asn Phe Ser His Arg Gly Ser Asp Thr Ala
Pro Ser Met 3300 3305 3310 Val Thr Ser Pro Gly Val Asp Thr Arg Ser
Gly Val Pro Thr Thr Thr 3315 3320 3325 Ile Pro Pro Ser Ile Pro Gly
Val Val Thr Ser Gln Val Thr Ser Ser 3330 3335 3340 Ala Thr Asp Thr
Ser Thr Ala Ile Pro Thr Leu Thr Pro Ser Pro Gly 3345 3350 3355 3360
Glu Pro Glu Thr Thr Ala Ser Ser Ala Thr His Pro Gly Thr Gln Thr
3365 3370 3375 Gly Phe Thr Val Pro Ile Arg Thr Val Pro Ser Ser Glu
Pro Asp Thr 3380 3385 3390 Met Ala Ser Trp Val Thr His Pro Pro Gln
Thr Ser Thr Pro Val Ser 3395 3400 3405 Arg Thr Thr Ser Ser Phe Ser
His Ser Ser Pro Asp Ala Thr Pro Val 3410 3415 3420 Met Ala Thr Ser
Pro Arg Thr Glu Ala Ser Ser Ala Val Leu Thr Thr 3425 3430 3435 3440
Ile Ser Pro Gly Ala Pro Glu Met Val Thr Ser Gln Ile Thr Ser Ser
3445 3450 3455 Gly Ala Ala Thr Ser Thr Thr Val Pro Thr Leu Thr His
Ser Pro Gly 3460 3465 3470 Met Pro Glu Thr Thr Ala Leu Leu Ser Thr
His Pro Arg Thr Glu Thr 3475 3480 3485 Ser Lys Thr Phe Pro Ala Ser
Thr Val Phe Pro Gln Val Ser Glu Thr 3490 3495 3500 Thr Ala Ser Leu
Thr Ile Arg Pro Gly Ala Glu Thr Ser Thr Ala Leu 3505 3510 3515 3520
Pro Thr Gln Thr Thr Ser Ser Leu Phe Thr Leu Leu Val Thr Gly Thr
3525 3530 3535 Ser Arg Val Asp Leu Ser Pro Thr Ala Ser Pro Gly Val
Ser Ala Lys 3540 3545 3550 Thr Ala Pro Leu Ser Thr His Pro Gly Thr
Glu Thr Ser Thr Met Ile 3555 3560 3565 Pro Thr Ser Thr Leu Ser Leu
Gly Leu Leu Glu Thr Thr Gly Leu Leu 3570 3575 3580 Ala Thr Ser Ser
Ser Ala Glu Thr Ser Thr Ser Thr Leu Thr Leu Thr 3585 3590 3595 3600
Val Ser Pro Ala Val Ser Gly Leu Ser Ser Ala Ser Ile Thr Thr Asp
3605 3610 3615 Lys Pro Gln Thr Val Thr Ser Trp Asn Thr Glu Thr Ser
Pro Ser Val 3620 3625 3630 Thr Ser Val Gly Pro Pro Glu Phe Ser Arg
Thr Val Thr Gly Thr Thr 3635 3640 3645 Met Thr Leu Ile Pro Ser Glu
Met Pro Thr Pro Pro Lys Thr Ser His 3650 3655 3660 Gly Glu Gly Val
Ser Pro Thr Thr Ile Leu Arg Thr Thr Met Val Glu 3665 3670 3675 3680
Ala Thr Asn Leu Ala Thr Thr Gly Ser Ser Pro Thr Val Ala Lys Thr
3685 3690 3695 Thr Thr Thr Phe Asn Thr Leu Ala Gly Ser Leu Phe Thr
Pro Leu Thr 3700 3705 3710 Thr Pro Gly Met Ser Thr Leu Ala Ser Glu
Ser Val Thr Ser Arg Thr 3715 3720 3725 Ser Tyr Asn His Arg Ser Trp
Ile Ser Thr Thr Ser Ser Tyr Asn Arg 3730 3735 3740
Arg Tyr Trp Thr Pro Ala Thr Ser Thr Pro Val Thr Ser Thr Phe Ser
3745 3750 3755 3760 Pro Gly Ile Ser Thr Ser Ser Ile Pro Ser Ser Thr
Ala Ala Thr Val 3765 3770 3775 Pro Phe Met Val Pro Phe Thr Leu Asn
Phe Thr Ile Thr Asn Leu Gln 3780 3785 3790 Tyr Glu Glu Asp Met Arg
His Pro Gly Ser Arg Lys Phe Asn Ala Thr 3795 3800 3805 Glu Arg Glu
Leu Gln Gly Leu Leu Lys Pro Leu Phe Arg Asn Ser Ser 3810 3815 3820
Leu Glu Tyr Leu Tyr Ser Gly Cys Arg Leu Ala Ser Leu Arg Pro Glu
3825 3830 3835 3840 Lys Asp Ser Ser Ala Thr Ala Val Asp Ala Ile Cys
Thr His Arg Pro 3845 3850 3855 Asp Pro Glu Asp Leu Gly Leu Asp Arg
Glu Arg Leu Tyr Trp Glu Leu 3860 3865 3870 Ser Asn Leu Thr Asn Gly
Ile Gln Glu Leu Gly Pro Tyr Thr Leu Asp 3875 3880 3885 Arg Asn Ser
Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Met Pro 3890 3895 3900
Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Val Gly Thr Ser Gly
3905 3910 3915 3920 Thr Pro Ser Ser Ser Pro Ser Pro Thr Thr Ala Gly
Pro Leu Leu Met 3925 3930 3935 Pro Phe Thr Leu Asn Phe Thr Ile Thr
Asn Leu Gln Tyr Glu Glu Asp 3940 3945 3950 Met Arg Arg Thr Gly Ser
Arg Lys Phe Asn Thr Met Glu Ser Val Leu 3955 3960 3965 Gln Gly Leu
Leu Lys Pro Leu Phe Lys Asn Thr Ser Val Gly Pro Leu 3970 3975 3980
Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Ala
3985 3990 3995 4000 Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Leu
Asp Pro Lys Ser 4005 4010 4015 Pro Gly Leu Asn Arg Glu Gln Leu Tyr
Trp Glu Leu Ser Lys Leu Thr 4020 4025 4030 Asn Asp Ile Glu Glu Leu
Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu 4035 4040 4045 Tyr Val Asn
Gly Phe Thr His Gln Ser Ser Val Ser Thr Thr Ser Thr 4050 4055 4060
Pro Gly Thr Ser Thr Val Asp Leu Arg Thr Ser Gly Thr Pro Ser Ser
4065 4070 4075 4080 Leu Ser Ser Pro Thr Ile Met Ala Ala Gly Pro Leu
Leu Val Pro Phe 4085 4090 4095 Thr Leu Asn Phe Thr Ile Thr Asn Leu
Gln Tyr Gly Glu Asp Met Gly 4100 4105 4110 His Pro Gly Ser Arg Lys
Phe Asn Thr Thr Glu Arg Val Leu Gln Gly 4115 4120 4125 Leu Leu Gly
Pro Ile Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser 4130 4135 4140
Gly Cys Arg Leu Thr Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr
4145 4150 4155 4160 Gly Val Asp Ala Ile Cys Ile His His Leu Asp Pro
Lys Ser Pro Gly 4165 4170 4175 Leu Asn Arg Glu Arg Leu Tyr Trp Glu
Leu Ser Gln Leu Thr Asn Gly 4180 4185 4190 Ile Lys Glu Leu Gly Pro
Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val 4195 4200 4205 Asn Gly Phe
Thr His Arg Thr Ser Val Pro Thr Thr Ser Thr Pro Gly 4210 4215 4220
Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Phe Ser Leu Pro
4225 4230 4235 4240 Ser Pro Ala Thr Ala Gly Pro Leu Leu Val Leu Phe
Thr Leu Asn Phe 4245 4250 4255 Thr Ile Thr Asn Leu Lys Tyr Glu Glu
Asp Met His Arg Pro Gly Ser 4260 4265 4270 Arg Lys Phe Asn Thr Thr
Glu Arg Val Leu Gln Thr Leu Val Gly Pro 4275 4280 4285 Met Phe Lys
Asn Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu 4290 4295 4300
Thr Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala
4305 4310 4315 4320 Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly
Val Asp Arg Glu 4325 4330 4335 Gln Leu Tyr Trp Glu Leu Ser Gln Leu
Thr Asn Gly Ile Lys Glu Leu 4340 4345 4350 Gly Pro Tyr Thr Leu Asp
Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr 4355 4360 4365 His Trp Ile
Pro Val Pro Thr Ser Ser Thr Pro Gly Thr Ser Thr Val 4370 4375 4380
Asp Leu Gly Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala
4385 4390 4395 4400 Thr Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn
Phe Thr Ile Thr 4405 4410 4415 Asn Leu Lys Tyr Glu Glu Asp Met His
Cys Pro Gly Ser Arg Lys Phe 4420 4425 4430 Asn Thr Thr Glu Arg Val
Leu Gln Ser Leu Leu Gly Pro Met Phe Lys 4435 4440 4445 Asn Thr Ser
Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu 4450 4455 4460
Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr
4465 4470 4475 4480 His Arg Leu Asp Pro Lys Ser Pro Gly Val Asp Arg
Glu Gln Leu Tyr 4485 4490 4495 Trp Glu Leu Ser Gln Leu Thr Asn Gly
Ile Lys Glu Leu Gly Pro Tyr 4500 4505 4510 Thr Leu Asp Arg Asn Ser
Leu Tyr Val Asn Gly Phe Thr His Gln Thr 4515 4520 4525 Ser Ala Pro
Asn Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly 4530 4535 4540
Thr Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala Gly Pro
4545 4550 4555 4560 Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr
Asn Leu Gln Tyr 4565 4570 4575 Glu Glu Asp Met His His Pro Gly Ser
Arg Lys Phe Asn Thr Thr Glu 4580 4585 4590 Arg Val Leu Gln Gly Leu
Leu Gly Pro Met Phe Lys Asn Thr Ser Val 4595 4600 4605 Gly Leu Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys 4610 4615 4620
Asn Gly Ala Ala Thr Gly Met Asp Ala Ile Cys Ser His Arg Leu Asp
4625 4630 4635 4640 Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr
Trp Glu Leu Ser 4645 4650 4655 Gln Leu Thr His Gly Ile Lys Glu Leu
Gly Pro Tyr Thr Leu Asp Arg 4660 4665 4670 Asn Ser Leu Tyr Val Asn
Gly Phe Thr His Arg Ser Ser Val Ala Pro 4675 4680 4685 Thr Ser Thr
Pro Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr 4690 4695 4700
Pro Ser Ser Leu Pro Ser Pro Thr Thr Ala Val Pro Leu Leu Val Pro
4705 4710 4715 4720 Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr
Gly Glu Asp Met 4725 4730 4735 Arg His Pro Gly Ser Arg Lys Phe Asn
Thr Thr Glu Arg Val Leu Gln 4740 4745 4750 Gly Leu Leu Gly Pro Leu
Phe Lys Asn Ser Ser Val Gly Pro Leu Tyr 4755 4760 4765 Ser Gly Cys
Arg Leu Ile Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala 4770 4775 4780
Thr Gly Val Asp Ala Ile Cys Thr His His Leu Asn Pro Gln Ser Pro
4785 4790 4795 4800 Gly Leu Asp Arg Glu Gln Leu Tyr Trp Gln Leu Ser
Gln Met Thr Asn 4805 4810 4815 Gly Ile Lys Glu Leu Gly Pro Tyr Thr
Leu Asp Arg Asn Ser Leu Tyr 4820 4825 4830 Val Asn Gly Phe Thr His
Arg Ser Ser Gly Leu Thr Thr Ser Thr Pro 4835 4840 4845 Trp Thr Ser
Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Ser Pro Val 4850 4855 4860
Pro Ser Pro Thr Thr Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn
4865 4870 4875 4880 Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met
His Arg Pro Gly 4885 4890 4895 Ser Arg Lys Phe Asn Ala Thr Glu Arg
Val Leu Gln Gly Leu Leu Ser 4900 4905 4910 Pro Ile Phe Lys Asn Ser
Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg 4915 4920 4925 Leu Thr Ser
Leu Arg Pro Glu Lys Asp Gly Ala Ala Thr Gly Met Asp 4930 4935 4940
Ala Val Cys Leu Tyr His Pro Asn Pro Lys Arg Pro Gly Leu Asp Arg
4945 4950 4955 4960 Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His
Asn Ile Thr Glu 4965 4970 4975 Leu Gly Pro Tyr Ser Leu Asp Arg Asp
Ser Leu Tyr Val Asn Gly Phe 4980 4985 4990 Thr His Gln Asn Ser Val
Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr 4995 5000 5005 Val Tyr Trp
Ala Thr Thr Gly Thr Pro Ser Ser Phe Pro Gly His Thr 5010 5015 5020
Glu Pro Gly Pro Leu Leu Ile Pro Phe Thr Phe Asn Phe Thr Ile Thr
5025 5030 5035 5040 Asn Leu His Tyr Glu Glu Asn Met Gln His Pro Gly
Ser Arg Lys Phe 5045 5050 5055 Asn Thr Thr Glu Arg Val Leu Gln Gly
Leu Leu Lys Pro Leu Phe Lys 5060 5065 5070 Asn Thr Ser Val Gly Pro
Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu
5075 5080 5085 Arg Pro Glu Lys Gln Glu Ala Ala Thr Gly Val Asp Thr
Ile Cys Thr 5090 5095 5100 His Arg Val Asp Pro Ile Gly Pro Gly Leu
Asp Arg Glu Arg Leu Tyr 5105 5110 5115 5120 Trp Glu Leu Ser Gln Leu
Thr Asn Ser Ile Thr Glu Leu Gly Pro Tyr 5125 5130 5135 Thr Leu Asp
Arg Asp Ser Leu Tyr Val Asn Gly Phe Asn Pro Trp Ser 5140 5145 5150
Ser Val Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr Val His Leu Ala
5155 5160 5165 Thr Ser Gly Thr Pro Ser Ser Leu Pro Gly His Thr Ala
Pro Val Pro 5170 5175 5180 Leu Leu Ile Pro Phe Thr Leu Asn Phe Thr
Ile Thr Asn Leu His Tyr 5185 5190 5195 5200 Glu Glu Asn Met Gln His
Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu 5205 5210 5215 Arg Val Leu
Gln Gly Leu Leu Lys Pro Leu Phe Lys Ser Thr Ser Val 5220 5225 5230
Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys
5235 5240 5245 His Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr Leu
Arg Leu Asp 5250 5255 5260 Pro Thr Gly Pro Gly Leu Asp Arg Glu Arg
Leu Tyr Trp Glu Leu Ser 5265 5270 5275 5280 Gln Leu Thr Asn Ser Val
Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg 5285 5290 5295 Asp Ser Leu
Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val Pro Thr 5300 5305 5310
Thr Ser Ile Pro Gly Thr Ser Ala Val His Leu Glu Thr Ser Gly Thr
5315 5320 5325 Pro Ala Ser Leu Pro Gly His Thr Ala Pro Gly Pro Leu
Leu Val Pro 5330 5335 5340 Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu
Gln Tyr Glu Glu Asp Met 5345 5350 5355 5360 Arg His Pro Gly Ser Arg
Lys Phe Asn Thr Thr Glu Arg Val Leu Gln 5365 5370 5375 Gly Leu Leu
Lys Pro Leu Phe Lys Ser Thr Ser Val Gly Pro Leu Tyr 5380 5385 5390
Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Arg Gly Ala Ala
5395 5400 5405 Thr Gly Val Asp Thr Ile Cys Thr His Arg Leu Asp Pro
Leu Asn Pro 5410 5415 5420 Gly Leu Asp Arg Glu Gln Leu Tyr Trp Glu
Leu Ser Lys Leu Thr Arg 5425 5430 5435 5440 Gly Ile Ile Glu Leu Gly
Pro Tyr Leu Leu Asp Arg Gly Ser Leu Tyr 5445 5450 5455 Val Asn Gly
Phe Thr His Arg Asn Phe Val Pro Ile Thr Ser Thr Pro 5460 5465 5470
Gly Thr Ser Thr Val His Leu Gly Thr Ser Glu Thr Pro Ser Ser Leu
5475 5480 5485 Pro Arg Pro Ile Val Pro Gly Pro Leu Leu Val Pro Phe
Thr Leu Asn 5490 5495 5500 Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu
Ala Met Arg His Pro Gly 5505 5510 5515 5520 Ser Arg Lys Phe Asn Thr
Thr Glu Arg Val Leu Gln Gly Leu Leu Arg 5525 5530 5535 Pro Leu Phe
Lys Asn Thr Ser Ile Gly Pro Leu Tyr Ser Ser Cys Arg 5540 5545 5550
Leu Thr Leu Leu Arg Pro Glu Lys Asp Lys Ala Ala Thr Arg Val Asp
5555 5560 5565 Ala Ile Cys Thr His His Pro Asp Pro Gln Ser Pro Gly
Leu Asn Arg 5570 5575 5580 Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu
Thr His Gly Ile Thr Glu 5585 5590 5595 5600 Leu Gly Pro Tyr Thr Leu
Asp Arg Asp Ser Leu Tyr Val Asp Gly Phe 5605 5610 5615 Thr His Trp
Ser Pro Ile Pro Thr Thr Ser Thr Pro Gly Thr Ser Ile 5620 5625 5630
Val Asn Leu Gly Thr Ser Gly Ile Pro Pro Ser Leu Pro Glu Thr Thr
5635 5640 5645 Ala Thr Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe
Thr Ile Thr 5650 5655 5660 Asn Leu Gln Tyr Glu Glu Asn Met Gly His
Pro Gly Ser Arg Lys Phe 5665 5670 5675 5680 Asn Ile Thr Glu Ser Val
Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys 5685 5690 5695 Ser Thr Ser
Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu 5700 5705 5710
Arg Pro Glu Lys Asp Gly Val Ala Thr Arg Val Asp Ala Ile Cys Thr
5715 5720 5725 His Arg Pro Asp Pro Lys Ile Pro Gly Leu Asp Arg Gln
Gln Leu Tyr 5730 5735 5740 Trp Glu Leu Ser Gln Leu Thr His Ser Ile
Thr Glu Leu Gly Pro Tyr 5745 5750 5755 5760 Thr Leu Asp Arg Asp Ser
Leu Tyr Val Asn Gly Phe Thr Gln Arg Ser 5765 5770 5775 Ser Val Pro
Thr Thr Ser Thr Pro Gly Thr Phe Thr Val Gln Pro Glu 5780 5785 5790
Thr Ser Glu Thr Pro Ser Ser Leu Pro Gly Pro Thr Ala Thr Gly Pro
5795 5800 5805 Val Leu Leu Pro Phe Thr Leu Asn Phe Thr Ile Ile Asn
Leu Gln Tyr 5810 5815 5820 Glu Glu Asp Met His Arg Pro Gly Ser Arg
Lys Phe Asn Thr Thr Glu 5825 5830 5835 5840 Arg Val Leu Gln Gly Leu
Leu Met Pro Leu Phe Lys Asn Thr Ser Val 5845 5850 5855 Ser Ser Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys 5860 5865 5870
Asp Gly Ala Ala Thr Arg Val Asp Ala Val Cys Thr His Arg Pro Asp
5875 5880 5885 Pro Lys Ser Pro Gly Leu Asp Arg Glu Arg Leu Tyr Trp
Lys Leu Ser 5890 5895 5900 Gln Leu Thr His Gly Ile Thr Glu Leu Gly
Pro Tyr Thr Leu Asp Arg 5905 5910 5915 5920 His Ser Leu Tyr Val Asn
Gly Phe Thr His Gln Ser Ser Met Thr Thr 5925 5930 5935 Thr Arg Thr
Pro Asp Thr Ser Thr Met His Leu Ala Thr Ser Arg Thr 5940 5945 5950
Pro Ala Ser Leu Ser Gly Pro Thr Thr Ala Ser Pro Leu Leu Val Leu
5955 5960 5965 Phe Thr Ile Asn Phe Thr Ile Thr Asn Leu Arg Tyr Glu
Glu Asn Met 5970 5975 5980 His His Pro Gly Ser Arg Lys Phe Asn Thr
Thr Glu Arg Val Leu Gln 5985 5990 5995 6000 Gly Leu Leu Arg Pro Val
Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr 6005 6010 6015 Ser Gly Cys
Arg Leu Thr Leu Leu Arg Pro Lys Lys Asp Gly Ala Ala 6020 6025 6030
Thr Lys Val Asp Ala Ile Cys Thr Tyr Arg Pro Asp Pro Lys Ser Pro
6035 6040 6045 Gly Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln
Leu Thr His 6050 6055 6060 Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu
Asp Arg Asp Ser Leu Tyr 6065 6070 6075 6080 Val Asn Gly Phe Thr Gln
Arg Ser Ser Val Pro Thr Thr Ser Ile Pro 6085 6090 6095 Gly Thr Pro
Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Val Ser Lys 6100 6105 6110
Pro Gly Pro Ser Ala Ala Ser Pro Leu Leu Val Leu Phe Thr Leu Asn
6115 6120 6125 Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met Gln
His Pro Gly 6130 6135 6140 Ser Arg Lys Phe Asn Thr Thr Glu Arg Val
Leu Gln Gly Leu Leu Arg 6145 6150 6155 6160 Ser Leu Phe Lys Ser Thr
Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg 6165 6170 6175 Leu Thr Leu
Leu Arg Pro Glu Lys Asp Gly Thr Ala Thr Gly Val Asp 6180 6185 6190
Ala Ile Cys Thr His His Pro Asp Pro Lys Ser Pro Arg Leu Asp Arg
6195 6200 6205 Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Asn
Ile Thr Glu 6210 6215 6220 Leu Gly Pro Tyr Ala Leu Asp Asn Asp Ser
Leu Phe Val Asn Gly Phe 6225 6230 6235 6240 Thr His Arg Ser Ser Val
Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr 6245 6250 6255 Val Tyr Leu
Gly Ala Ser Lys Thr Pro Ala Ser Ile Phe Gly Pro Ser 6260 6265 6270
Ala Ala Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr
6275 6280 6285 Asn Leu Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg
Lys Phe Asn 6290 6295 6300 Thr Thr Glu Arg Val Leu Gln Gly Leu Leu
Arg Pro Leu Phe Lys Asn 6305 6310 6315 6320 Thr Ser Val Gly Pro Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg 6325 6330 6335 Pro Glu Lys
Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr His 6340 6345 6350
Arg Pro Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu Tyr Leu
6355 6360 6365 Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly
Pro Tyr Thr 6370 6375 6380 Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly
Phe Thr His Arg Ser Ser 6385 6390 6395 6400 Val Pro Thr Thr Ser Thr
Gly Val Val Ser Glu Glu Pro Phe Thr Leu 6405 6410 6415
Asn Phe Thr Ile Asn Asn Leu Arg Tyr Met Ala Asp Met Gly Gln Pro
6420 6425 6430 Gly Ser Leu Lys Phe Asn Ile Thr Asp Asn Val Met Gln
His Leu Leu 6435 6440 6445 Ser Pro Leu Phe Gln Arg Ser Ser Leu Gly
Ala Arg Tyr Thr Gly Cys 6450 6455 6460 Arg Val Ile Ala Leu Arg Ser
Val Lys Asn Gly Ala Glu Thr Arg Val 6465 6470 6475 6480 Asp Leu Leu
Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro 6485 6490 6495
Ile Lys Gln Val Phe His Glu Leu Ser Gln Gln Thr His Gly Ile Thr
6500 6505 6510 Arg Leu Gly Pro Tyr Ser Leu Asp Lys Asp Ser Leu Tyr
Leu Asn Gly 6515 6520 6525 Tyr Asn Glu Pro Gly Pro Asp Glu Pro Pro
Thr Thr Pro Lys Pro Ala 6530 6535 6540 Thr Thr Phe Leu Pro Pro Leu
Ser Glu Ala Thr Thr Ala Met Gly Tyr 6545 6550 6555 6560 His Leu Lys
Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn Leu Gln Tyr 6565 6570 6575
Ser Pro Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu Gly
6580 6585 6590 Val Leu Gln His Leu Leu Arg Pro Leu Phe Gln Lys Ser
Ser Met Gly 6595 6600 6605 Pro Phe Tyr Leu Gly Cys Gln Leu Ile Ser
Leu Arg Pro Glu Lys Asp 6610 6615 6620 Gly Ala Ala Thr Gly Val Asp
Thr Thr Cys Thr Tyr His Pro Asp Pro 6625 6630 6635 6640 Val Gly Pro
Gly Leu Asp Ile Gln Gln Leu Tyr Trp Glu Leu Ser Gln 6645 6650 6655
Leu Thr His Gly Val Thr Gln Leu Gly Phe Tyr Val Leu Asp Arg Asp
6660 6665 6670 Ser Leu Phe Ile Asn Gly Tyr Ala Pro Gln Asn Leu Ser
Ile Arg Gly 6675 6680 6685 Glu Tyr Gln Ile Asn Phe His Ile Val Asn
Trp Asn Leu Ser Asn Pro 6690 6695 6700 Asp Pro Thr Ser Ser Glu Tyr
Ile Thr Leu Leu Arg Asp Ile Gln Asp 6705 6710 6715 6720 Lys Val Thr
Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr Phe Arg 6725 6730 6735
Phe Cys Leu Val Thr Asn Leu Thr Met Asp Ser Val Leu Val Thr Val
6740 6745 6750 Lys Ala Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val
Glu Gln Val 6755 6760 6765 Phe Leu Asp Lys Thr Leu Asn Ala Ser Phe
His Trp Leu Gly Ser Thr 6770 6775 6780 Tyr Gln Leu Val Asp Ile His
Val Thr Glu Met Glu Ser Ser Val Tyr 6785 6790 6795 6800 Gln Pro Thr
Ser Ser Ser Ser Thr Gln His Phe Tyr Leu Asn Phe Thr 6805 6810 6815
Ile Thr Asn Leu Pro Tyr Ser Gln Asp Lys Ala Gln Pro Gly Thr Thr
6820 6825 6830 Asn Tyr Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu
Asn Gln Leu 6835 6840 6845 Phe Arg Asn Ser Ser Ile Lys Ser Tyr Phe
Ser Asp Cys Gln Val Ser 6850 6855 6860 Thr Phe Arg Ser Val Pro Asn
Arg His His Thr Gly Val Asp Ser Leu 6865 6870 6875 6880 Cys Asn Phe
Ser Pro Leu Ala Arg Arg Val Asp Arg Val Ala Ile Tyr 6885 6890 6895
Glu Glu Phe Leu Arg Met Thr Arg Asn Gly Thr Gln Leu Gln Asn Phe
6900 6905 6910 Thr Leu Asp Arg Ser Ser Val Leu Val Asp Gly Tyr Ser
Pro Asn Arg 6915 6920 6925 Asn Glu Pro Leu Thr Gly Asn Ser Asp Leu
Pro Phe Trp Ala Val Ile 6930 6935 6940 Leu Ile Gly Leu Ala Gly Leu
Leu Gly Leu Ile Thr Cys Leu Ile Cys 6945 6950 6955 6960 Gly Val Leu
Val Thr Thr Arg Arg Arg Lys Lys Glu Gly Glu Tyr Asn 6965 6970 6975
Val Gln Gln Gln Cys Pro Gly Tyr Tyr Gln Ser His Leu Asp Leu Glu
6980 6985 6990 Asp Leu Gln 6995 <210> SEQ ID NO 5 <211>
LENGTH: 622 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <223> OTHER INFORMATION: MSLN
<400> SEQUENCE: 5 Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly
Ser Cys Gly Thr Pro 1 5 10 15 Ala Leu Gly Ser Leu Leu Phe Leu Leu
Phe Ser Leu Gly Trp Val Gln 20 25 30 Pro Ser Arg Thr Leu Ala Gly
Glu Thr Gly Gln Glu Ala Ala Pro Leu 35 40 45 Asp Gly Val Leu Ala
Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg 50 55 60 Gln Leu Leu
Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu 65 70 75 80 Arg
Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu 85 90
95 Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro
100 105 110 Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu
Asn Pro 115 120 125 Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe
Phe Ser Arg Ile 130 135 140 Thr Lys Ala Asn Val Asp Leu Leu Pro Arg
Gly Ala Pro Glu Arg Gln 145 150 155 160 Arg Leu Leu Pro Ala Ala Leu
Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170 175 Leu Ser Glu Ala Asp
Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185 190 Pro Gly Arg
Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu 195 200 205 Val
Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg 210 215
220 Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp
225 230 235 240 Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro
Val Leu Gly 245 250 255 Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile
Val Ala Ala Trp Arg 260 265 270 Gln Arg Ser Ser Arg Asp Pro Ser Trp
Arg Gln Pro Glu Arg Thr Ile 275 280 285 Leu Arg Pro Arg Phe Arg Arg
Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295 300 Gly Lys Lys Ala Arg
Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys 305 310 315 320 Trp Glu
Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met 325 330 335
Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu 340
345 350 Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser
Val 355 360 365 Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro
Glu Asp Ile 370 375 380 Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu
Lys Ala Leu Leu Glu 385 390 395 400 Val Asn Lys Gly His Glu Met Ser
Pro Gln Val Ala Thr Leu Ile Asp 405 410 415 Arg Phe Val Lys Gly Arg
Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr 420 425 430 Leu Thr Ala Phe
Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu 435 440 445 Leu Ser
Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp 450 455 460
Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala 465
470 475 480 Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val
Lys Ile 485 490 495 Gln Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu
Lys Ala Leu Ser 500 505 510 Gln Gln Asn Val Ser Met Asp Leu Ala Thr
Phe Met Lys Leu Arg Thr 515 520 525 Asp Ala Val Leu Pro Leu Thr Val
Ala Glu Val Gln Lys Leu Leu Gly 530 535 540 Pro His Val Glu Gly Leu
Lys Ala Glu Glu Arg His Arg Pro Val Arg 545 550 555 560 Asp Trp Ile
Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu 565 570 575 Gly
Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser 580 585
590 Met Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu Gly Pro Gly Pro
595 600 605 Val Leu Thr Val Leu Ala Leu Leu Leu Ala Ser Thr Leu Ala
610 615 620 <210> SEQ ID NO 6 <211> LENGTH: 690
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: SLC34A2
<400> SEQUENCE: 6 Met Ala Pro Trp Pro Glu Leu Gly Asp Ala Gln
Pro Asn Pro Asp Lys 1 5 10 15 Tyr Leu Glu Gly Ala Ala Gly Gln Gln
Pro Thr Ala Pro Asp Lys Ser
20 25 30 Lys Glu Thr Asn Lys Thr Asp Asn Thr Glu Ala Pro Val Thr
Lys Ile 35 40 45 Glu Leu Leu Pro Ser Tyr Ser Thr Ala Thr Leu Ile
Asp Glu Pro Thr 50 55 60 Glu Val Asp Asp Pro Trp Asn Leu Pro Thr
Leu Gln Asp Ser Gly Ile 65 70 75 80 Lys Trp Ser Glu Arg Asp Thr Lys
Gly Lys Ile Leu Cys Phe Phe Gln 85 90 95 Gly Ile Gly Arg Leu Ile
Leu Leu Leu Gly Phe Leu Tyr Phe Phe Val 100 105 110 Cys Ser Leu Asp
Ile Leu Ser Ser Ala Phe Gln Leu Val Gly Gly Lys 115 120 125 Met Ala
Gly Gln Phe Phe Ser Asn Ser Ser Ile Met Ser Asn Pro Leu 130 135 140
Leu Gly Leu Val Ile Gly Val Leu Val Thr Val Leu Val Gln Ser Ser 145
150 155 160 Ser Thr Ser Thr Ser Ile Val Val Ser Met Val Ser Ser Ser
Leu Leu 165 170 175 Thr Val Arg Ala Ala Ile Pro Ile Ile Met Gly Ala
Asn Ile Gly Thr 180 185 190 Ser Ile Thr Asn Thr Ile Val Ala Leu Met
Gln Val Gly Asp Arg Ser 195 200 205 Glu Phe Arg Arg Ala Phe Ala Gly
Ala Thr Val His Asp Phe Phe Asn 210 215 220 Trp Leu Ser Val Leu Val
Leu Leu Pro Val Glu Val Ala Thr His Tyr 225 230 235 240 Leu Glu Ile
Ile Thr Gln Leu Ile Val Glu Ser Phe His Phe Lys Asn 245 250 255 Gly
Glu Asp Ala Pro Asp Leu Leu Lys Val Ile Thr Lys Pro Phe Thr 260 265
270 Lys Leu Ile Val Gln Leu Asp Lys Lys Val Ile Ser Gln Ile Ala Met
275 280 285 Asn Asp Glu Lys Ala Lys Asn Lys Ser Leu Val Lys Ile Trp
Cys Lys 290 295 300 Thr Phe Thr Asn Lys Thr Gln Ile Asn Val Thr Val
Pro Ser Thr Ala 305 310 315 320 Asn Cys Thr Ser Pro Ser Leu Cys Trp
Thr Asp Gly Ile Gln Asn Trp 325 330 335 Thr Met Lys Asn Val Thr Tyr
Lys Glu Asn Ile Ala Lys Cys Gln His 340 345 350 Ile Phe Val Asn Phe
His Leu Pro Asp Leu Ala Val Gly Thr Ile Leu 355 360 365 Leu Ile Leu
Ser Leu Leu Val Leu Cys Gly Cys Leu Ile Met Ile Val 370 375 380 Lys
Ile Leu Gly Ser Val Leu Lys Gly Gln Val Ala Thr Val Ile Lys 385 390
395 400 Lys Thr Ile Asn Thr Asp Phe Pro Phe Pro Phe Ala Trp Leu Thr
Gly 405 410 415 Tyr Leu Ala Ile Leu Val Gly Ala Gly Met Thr Phe Ile
Val Gln Ser 420 425 430 Ser Ser Val Phe Thr Ser Ala Leu Thr Pro Leu
Ile Gly Ile Gly Val 435 440 445 Ile Thr Ile Glu Arg Ala Tyr Pro Leu
Thr Leu Gly Ser Asn Ile Gly 450 455 460 Thr Thr Thr Thr Ala Ile Leu
Ala Ala Leu Ala Ser Pro Gly Asn Ala 465 470 475 480 Leu Arg Ser Ser
Leu Gln Ile Ala Leu Cys His Phe Phe Phe Asn Ile 485 490 495 Ser Gly
Ile Leu Leu Trp Tyr Pro Ile Pro Phe Thr Arg Leu Pro Ile 500 505 510
Arg Met Ala Lys Gly Leu Gly Asn Ile Ser Ala Lys Tyr Arg Trp Phe 515
520 525 Ala Val Phe Tyr Leu Ile Ile Phe Phe Phe Leu Ile Pro Leu Thr
Val 530 535 540 Phe Gly Leu Ser Leu Ala Gly Trp Arg Val Leu Val Gly
Val Gly Val 545 550 555 560 Pro Val Val Phe Ile Ile Ile Leu Val Leu
Cys Leu Arg Leu Leu Gln 565 570 575 Ser Arg Cys Pro Arg Val Leu Pro
Lys Lys Leu Gln Asn Trp Asn Phe 580 585 590 Leu Pro Leu Trp Met Arg
Ser Leu Lys Pro Trp Asp Ala Val Val Ser 595 600 605 Lys Phe Thr Gly
Cys Phe Gln Met Arg Cys Cys Tyr Cys Cys Arg Val 610 615 620 Cys Cys
Arg Ala Cys Cys Leu Leu Cys Gly Cys Pro Lys Cys Cys Arg 625 630 635
640 Cys Ser Lys Cys Cys Glu Asp Leu Glu Glu Ala Gln Glu Gly Gln Asp
645 650 655 Val Pro Val Lys Ala Pro Glu Thr Phe Asp Asn Ile Thr Ile
Ser Arg 660 665 670 Glu Ala Gln Gly Glu Val Pro Ala Ser Asp Ser Lys
Thr Glu Cys Thr 675 680 685 Ala Leu 690 <210> SEQ ID NO 7
<211> LENGTH: 1093 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: KIAA1445 <400> SEQUENCE: 7 Met Val Leu Ala Gly
Pro Leu Ala Val Ser Leu Leu Leu Pro Ser Leu 1 5 10 15 Thr Leu Leu
Val Ser His Leu Ser Ser Ser Gln Asp Val Ser Ser Glu 20 25 30 Pro
Ser Ser Glu Gln Gln Leu Cys Ala Leu Ser Lys His Pro Thr Val 35 40
45 Ala Phe Glu Asp Leu Gln Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly
50 55 60 Ala Arg Asp Phe Ser Gln Leu Ala Leu Asp Pro Ser Gly Asn
Gln Leu 65 70 75 80 Ile Val Gly Ala Arg Asn Tyr Leu Phe Arg Leu Ser
Leu Ala Asn Val 85 90 95 Ser Leu Leu Gln Ala Thr Glu Trp Ala Ser
Ser Glu Asp Thr Arg Arg 100 105 110 Ser Cys Gln Ser Lys Gly Lys Thr
Glu Glu Glu Cys Gln Asn Tyr Val 115 120 125 Arg Val Leu Ile Val Ala
Gly Arg Lys Val Phe Met Cys Gly Thr Asn 130 135 140 Ala Phe Ser Pro
Met Cys Thr Ser Arg Gln Val Gly Asn Leu Ser Arg 145 150 155 160 Thr
Thr Glu Lys Ile Asn Gly Val Ala Arg Cys Pro Tyr Asp Pro Arg 165 170
175 His Asn Ser Thr Ala Val Ile Ser Ser Gln Gly Glu Leu Tyr Ala Ala
180 185 190 Thr Val Ile Asp Phe Ser Gly Arg Asp Pro Ala Ile Tyr Arg
Ser Leu 195 200 205 Gly Ser Gly Pro Pro Leu Arg Thr Ala Gln Tyr Asn
Ser Lys Trp Leu 210 215 220 Asn Glu Pro Asn Phe Val Ala Ala Tyr Asp
Ile Gly Leu Phe Ala Tyr 225 230 235 240 Phe Phe Leu Arg Glu Asn Ala
Val Glu His Asp Cys Gly Arg Thr Val 245 250 255 Tyr Ser Arg Val Ala
Arg Val Cys Lys Asn Asp Val Gly Gly Arg Phe 260 265 270 Leu Leu Glu
Asp Thr Trp Thr Thr Phe Met Lys Ala Arg Leu Asn Cys 275 280 285 Ser
Arg Pro Gly Glu Val Pro Phe Tyr Tyr Asn Glu Leu Gln Ser Ala 290 295
300 Phe His Leu Pro Glu Gln Asp Leu Ile Tyr Gly Val Phe Thr Thr Asn
305 310 315 320 Val Asn Ser Ile Ala Ala Ser Ala Val Cys Ala Phe Asn
Leu Ser Ala 325 330 335 Ile Ser Gln Ala Phe Asn Gly Pro Phe Arg Tyr
Gln Glu Asn Pro Arg 340 345 350 Ala Ala Trp Leu Pro Ile Ala Asn Pro
Ile Pro Asn Phe Gln Cys Gly 355 360 365 Thr Leu Pro Glu Thr Gly Pro
Asn Glu Asn Leu Thr Glu Arg Ser Leu 370 375 380 Gln Asp Ala Gln Arg
Leu Phe Leu Met Ser Glu Ala Val Gln Pro Val 385 390 395 400 Thr Pro
Glu Pro Cys Val Thr Gln Asp Ser Val Arg Phe Ser His Leu 405 410 415
Val Val Asp Leu Val Gln Ala Lys Asp Thr Leu Tyr His Val Leu Tyr 420
425 430 Ile Gly Thr Glu Ser Gly Thr Ile Leu Lys Ala Leu Ser Thr Ala
Ser 435 440 445 Arg Ser Leu His Gly Cys Tyr Leu Glu Glu Leu His Val
Leu Pro Pro 450 455 460 Gly Arg Arg Glu Pro Leu Arg Ser Leu Arg Ile
Leu His Ser Ala Arg 465 470 475 480 Ala Leu Phe Val Gly Leu Arg Asp
Gly Val Leu Arg Val Pro Leu Glu 485 490 495 Arg Cys Ala Ala Tyr Arg
Ser Gln Gly Ala Cys Leu Gly Ala Arg Asp 500 505 510 Pro Tyr Cys Gly
Trp Asp Gly Lys Gln Gln Arg Cys Ser Thr Leu Glu 515 520 525 Asp Ser
Ser Asn Met Ser Leu Trp Thr Gln Asn Ile Thr Ala Cys Pro 530 535 540
Val Arg Asn Val Thr Arg Asp Gly Gly Phe Gly Pro Trp Ser Pro Trp 545
550 555 560 Gln Pro Cys Glu His Leu Asp Gly Asp Asn Ser Gly Ser Cys
Leu Cys 565 570 575 Arg Ala Arg Ser Cys Asp Ser Pro Arg Pro Arg Cys
Gly Gly Leu Asp 580 585 590 Cys Leu Gly Pro Ala Ile His Ile Ala Asn
Cys Ser Arg Asn Gly Ala 595 600 605 Trp Thr Pro Trp Ser Ser Trp Ala
Leu Cys Ser Thr Ser Cys Gly Ile 610 615 620 Gly Phe Gln Val Arg Gln
Arg Ser Cys Ser Asn Pro Ala Pro Arg His 625 630 635 640 Gly Gly Arg
Ile Cys Val Gly Lys Ser Arg Glu Glu Arg Phe Cys Asn 645 650 655 Glu
Asn Thr Pro Cys Pro Val Pro Ile Phe Trp Ala Ser Trp Gly Ser 660 665
670 Trp Ser Lys Cys Ser Ser Asn Cys Gly Gly Gly Met Gln Ser Arg Arg
675 680 685 Arg Ala Cys Glu Asn Gly Asn Ser Cys Leu Gly Cys Gly Val
Glu Phe 690 695 700 Lys Thr Cys Asn Pro Glu Gly Cys Pro Glu Val Arg
Arg Asn Thr Pro 705 710 715 720 Trp Thr Pro Trp Leu Pro Val Asn Val
Thr Gln Gly Gly Ala Arg Gln 725 730 735 Glu Gln Arg Phe Arg Phe Thr
Cys Arg Ala Pro Leu Ala Asp Pro His 740 745 750 Gly Leu Gln Phe Gly
Arg Arg Arg Thr Glu Thr Arg Thr Cys Pro Ala 755 760 765 Asp Gly Ser
Gly Ser Cys Asp Thr Asp Ala Leu Val Glu Asp Leu Leu 770 775 780
Arg Ser Gly Ser Thr Ser Pro His Thr Val Ser Gly Gly Trp Ala Ala 785
790 795 800 Trp Gly Pro Trp Ser Ser Cys Ser Arg Asp Cys Glu Leu Gly
Phe Arg 805 810 815 Val Arg Lys Arg Thr Cys Thr Asn Pro Glu Pro Arg
Asn Gly Gly Leu 820 825 830 Pro Cys Val Gly Asp Ala Ala Glu Tyr Gln
Asp Cys Asn Pro Gln Ala 835 840 845 Cys Pro Val Arg Gly Ala Trp Ser
Cys Trp Thr Ser Trp Ser Pro Cys 850 855 860 Ser Ala Ser Cys Gly Gly
Gly His Tyr Gln Arg Thr Arg Ser Cys Thr 865 870 875 880 Ser Pro Ala
Pro Ser Pro Gly Glu Asp Ile Cys Leu Gly Leu His Thr 885 890 895 Glu
Glu Ala Leu Cys Ala Thr Gln Ala Cys Pro Glu Gly Trp Ser Pro 900 905
910 Trp Ser Glu Trp Ser Lys Cys Thr Asp Asp Gly Ala Gln Ser Arg Ser
915 920 925 Arg His Cys Glu Glu Leu Leu Pro Gly Ser Ser Ala Cys Ala
Gly Asn 930 935 940 Ser Ser Gln Ser Arg Pro Cys Pro Tyr Ser Glu Ile
Pro Val Ile Leu 945 950 955 960 Pro Ala Ser Ser Met Glu Glu Ala Thr
Gly Cys Ala Gly Phe Asn Leu 965 970 975 Ile His Leu Val Ala Thr Gly
Ile Ser Cys Phe Leu Gly Ser Gly Leu 980 985 990 Leu Thr Leu Ala Val
Tyr Leu Ser Cys Gln His Cys Gln Arg Gln Ser 995 1000 1005 Gln Glu
Ser Thr Leu Val His Pro Ala Thr Pro Asn His Leu His Tyr 1010 1015
1020 Lys Gly Gly Gly Thr Pro Lys Asn Glu Lys Tyr Thr Pro Met Glu
Phe 1025 1030 1035 1040 Lys Thr Leu Asn Lys Asn Asn Leu Ile Pro Asp
Asp Arg Ala Asn Phe 1045 1050 1055 Tyr Pro Leu Gln Gln Thr Asn Val
Tyr Thr Thr Thr Tyr Tyr Pro Ser 1060 1065 1070 Pro Leu Asn Lys His
Ser Phe Arg Pro Glu Ala Ser Pro Gly Gln Arg 1075 1080 1085 Cys Phe
Pro Asn Ser 1090 <210> SEQ ID NO 8 <211> LENGTH: 141
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: PSCA hlg
<400> SEQUENCE: 8 Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys
Gly Leu Phe Leu Leu 1 5 10 15 Pro Gly Phe Ala Leu Gln Ile Gln Cys
Tyr Gln Cys Glu Glu Phe Gln 20 25 30 Leu Asn Asn Asp Cys Ser Ser
Pro Glu Phe Ile Val Asn Cys Thr Val 35 40 45 Asn Val Gln Asp Met
Cys Gln Lys Glu Val Met Glu Gln Ser Ala Gly 50 55 60 Ile Met Tyr
Arg Lys Ser Cys Ala Ser Ser Ala Ala Cys Leu Ile Ala 65 70 75 80 Ser
Ala Gly Tyr Gln Ser Phe Cys Ser Pro Gly Lys Leu Asn Ser Val 85 90
95 Cys Ile Ser Cys Cys Asn Thr Pro Leu Cys Asn Gly Pro Arg Pro Lys
100 105 110 Lys Arg Gly Ser Ser Ala Ser Ala Leu Arg Pro Gly Leu Arg
Thr Thr 115 120 125 Ile Leu Phe Leu Lys Leu Ala Leu Phe Ser Ala His
Cys 130 135 140 <210> SEQ ID NO 9 <211> LENGTH: 442
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: ETBR
<400> SEQUENCE: 9 Met Gln Pro Pro Pro Ser Leu Cys Gly Arg Ala
Leu Val Ala Leu Val 1 5 10 15 Leu Ala Cys Gly Leu Ser Arg Ile Trp
Gly Glu Glu Arg Gly Phe Pro 20 25 30 Pro Asp Arg Ala Thr Pro Leu
Leu Gln Thr Ala Glu Ile Met Thr Pro 35 40 45 Pro Thr Lys Thr Leu
Trp Pro Lys Gly Ser Asn Ala Ser Leu Ala Arg 50 55 60 Ser Leu Ala
Pro Ala Glu Val Pro Lys Gly Asp Arg Thr Ala Gly Ser 65 70 75 80 Pro
Pro Arg Thr Ile Ser Pro Pro Pro Cys Gln Gly Pro Ile Glu Ile 85 90
95 Lys Glu Thr Phe Lys Tyr Ile Asn Thr Val Val Ser Cys Leu Val Phe
100 105 110 Val Leu Gly Ile Ile Gly Asn Ser Thr Leu Leu Arg Ile Ile
Tyr Lys 115 120 125 Asn Lys Cys Met Arg Asn Gly Pro Asn Ile Leu Ile
Ala Ser Leu Ala 130 135 140 Leu Gly Asp Leu Leu His Ile Val Ile Asp
Ile Pro Ile Asn Val Tyr 145 150 155 160 Lys Leu Leu Ala Glu Asp Trp
Pro Phe Gly Ala Glu Met Cys Lys Leu 165 170 175 Val Pro Phe Ile Gln
Lys Ala Ser Val Gly Ile Thr Val Leu Ser Leu 180 185 190 Cys Ala Leu
Ser Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg 195 200 205 Ile
Lys Gly Ile Gly Val Pro Lys Trp Thr Ala Val Glu Ile Val Leu 210 215
220 Ile Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala Ile Gly Phe
225 230 235 240 Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser Tyr Leu Arg
Ile Cys Leu 245 250 255 Leu His Pro Val Gln Lys Thr Ala Phe Met Gln
Phe Tyr Lys Thr Ala 260 265 270 Lys Asp Trp Trp Leu Phe Ser Phe Tyr
Phe Cys Leu Pro Leu Ala Ile 275 280 285 Thr Ala Phe Phe Tyr Thr Leu
Met Thr Cys Glu Met Leu Arg Lys Lys 290 295 300 Ser Gly Met Gln Ile
Ala Leu Asn Asp His Leu Lys Gln Arg Arg Glu 305 310 315 320 Val Ala
Lys Thr Val Phe Cys Leu Val Leu Val Phe Ala Leu Cys Trp 325 330 335
Leu Pro Leu His Leu Ser Arg Ile Leu Lys Leu Thr Leu Tyr Asn Gln 340
345 350 Asn Asp Pro Asn Arg Cys Glu Leu Leu Ser Phe Leu Leu Val Leu
Asp 355 360 365 Tyr Ile Gly Ile Asn Met Ala Ser Leu Asn Ser Cys Ile
Asn Pro Ile 370 375 380 Ala Leu Tyr Leu Val Ser Lys Arg Phe Lys Asn
Cys Phe Lys Ser Cys 385 390 395 400 Leu Cys Cys Trp Cys Gln Ser Phe
Glu Glu Lys Gln Ser Leu Glu Glu 405 410 415 Lys Gln Ser Cys Leu Lys
Phe Lys Ala Asn Asp His Gly Tyr Asp Asn 420 425 430 Phe Arg Ser Ser
Asn Lys Tyr Ser Ser Ser 435 440 <210> SEQ ID NO 10
<211> LENGTH: 783 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
RNF124 <400> SEQUENCE: 10 Met Ser Gly Gly His Gln Leu Gln Leu
Ala Ala Leu Trp Pro Trp Leu 1 5 10 15 Leu Met Ala Thr Leu Gln Ala
Gly Phe Gly Arg Thr Gly Leu Val Leu 20 25 30 Ala Ala Ala Val Glu
Ser Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile 35 40 45 Arg Val Ile
Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn Leu Thr 50 55 60 Leu
Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala Glu Gly 65 70
75 80 Lys Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn Ala Ser Asp
Asp 85 90 95 Asp Asn Leu Glu Pro Gly Phe Ile Ser Ile Val Lys Leu
Glu Ser Pro 100 105 110 Arg Arg Ala Pro Arg Pro Cys Leu Ser Leu Ala
Ser Lys Ala Arg Met 115 120 125 Ala Gly Glu Arg Gly Ala Ser Ala Val
Leu Phe Asp Ile Thr Glu Asp 130 135 140 Arg Ala Ala Ala Glu Gln Leu
Gln Gln Pro Leu Gly Leu Thr Trp Pro 145 150 155 160 Val Val Leu Ile
Trp Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val 165 170 175 Tyr Lys
Asn Gln Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro 180 185 190
Ala Trp Pro Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr 195
200 205 Ile Phe Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg
Pro 210 215 220 Arg His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala
Trp Ala Ile 225 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala
Ser Cys Arg Gln Ala Arg 245 250 255 Gly Glu Trp Pro Asp Ser Gly Ser
Ser Cys Ser Ser Ala Pro Val Cys 260 265 270 Ala Ile Cys Leu Glu Glu
Phe Ser Glu Gly Gln Glu Leu Arg Val Ile 275 280 285 Ser Cys Leu His
Glu Phe His Arg Asn Cys Val Asp Pro Trp Leu His 290 295 300 Gln His
Arg Thr Cys Pro Leu Cys Val Phe Asn Ile Thr Glu Gly Asp 305 310 315
320 Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg Ser Tyr Gln Glu Pro Gly
325 330 335 Arg Arg Leu His Leu Ile Arg Gln His Pro Gly His Ala His
Tyr His 340 345 350 Leu Pro Ala Ala Tyr Leu Leu Gly Pro Ser Arg Ser
Ala Val Ala Arg 355 360 365 Pro Pro Arg Pro Gly Pro Phe Leu Pro Ser
Gln Glu Pro Gly Met Gly 370 375 380 Pro Arg His His Arg Phe Pro Arg
Ala Ala His Pro Arg Ala Pro Gly
385 390 395 400 Glu Gln Gln Arg Leu Ala Gly Ala Gln His Pro Tyr Ala
Gln Gly Trp 405 410 415 Gly Met Ser His Leu Gln Ser Thr Ser Gln His
Pro Ala Ala Cys Pro 420 425 430 Val Pro Leu Arg Arg Ala Arg Pro Pro
Asp Ser Ser Gly Ser Gly Glu 435 440 445 Ser Tyr Cys Thr Glu Arg Ser
Gly Tyr Leu Ala Asp Gly Pro Ala Ser 450 455 460 Asp Ser Ser Ser Gly
Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 465 470 475 480 Asn Cys
Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser Thr 485 490 495
Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr Cys Ser 500
505 510 Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser Val Thr
Ser 515 520 525 Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly Glu
Thr Gln Val 530 535 540 Ser Ser His Val His Tyr His Arg His Arg His
His His Tyr Lys Lys 545 550 555 560 Arg Phe Gln Trp His Gly Arg Lys
Pro Gly Pro Glu Thr Gly Val Pro 565 570 575 Gln Ser Arg Pro Pro Ile
Pro Arg Thr Gln Pro Gln Pro Glu Pro Pro 580 585 590 Ser Pro Asp Gln
Gln Val Thr Gly Ser Asn Ser Ala Ala Pro Ser Gly 595 600 605 Arg Leu
Ser Asn Pro Gln Cys Pro Arg Ala Leu Pro Glu Pro Ala Pro 610 615 620
Gly Pro Val Asp Ala Ser Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe 625
630 635 640 Asn Leu Gln Lys Ser Ser Leu Ser Ala Arg His Pro Gln Arg
Lys Arg 645 650 655 Arg Gly Gly Pro Ser Glu Pro Thr Pro Gly Ser Arg
Pro Gln Asp Ala 660 665 670 Thr Val His Pro Ala Cys Gln Ile Phe Pro
His Tyr Thr Pro Ser Val 675 680 685 Ala Tyr Pro Trp Ser Pro Glu Ala
His Pro Leu Ile Cys Gly Pro Pro 690 695 700 Gly Leu Asp Lys Arg Leu
Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 705 710 715 720 Asn Ser Gln
Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu Glu 725 730 735 Pro
His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp Thr Ala 740 745
750 Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu Ser Ala Gln
755 760 765 Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala
Val 770 775 780 <210> SEQ ID NO 11 <211> LENGTH: 490
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: STEAP2
<400> SEQUENCE: 11 Met Glu Ser Ile Ser Met Met Gly Ser Pro
Lys Ser Leu Ser Glu Thr 1 5 10 15 Val Leu Pro Asn Gly Ile Asn Gly
Ile Lys Asp Ala Arg Lys Val Thr 20 25 30 Val Gly Val Ile Gly Ser
Gly Asp Phe Ala Lys Ser Leu Thr Ile Arg 35 40 45 Leu Ile Arg Cys
Gly Tyr His Val Val Ile Gly Ser Arg Asn Pro Lys 50 55 60 Phe Ala
Ser Glu Phe Phe Pro His Val Val Asp Val Thr His His Glu 65 70 75 80
Asp Ala Leu Thr Lys Thr Asn Ile Ile Phe Val Ala Ile His Arg Glu 85
90 95 His Tyr Thr Ser Leu Trp Asp Leu Arg His Leu Leu Val Gly Lys
Ile 100 105 110 Leu Ile Asp Val Ser Asn Asn Met Arg Ile Asn Gln Tyr
Pro Glu Ser 115 120 125 Asn Ala Glu Tyr Leu Ala Ser Leu Phe Pro Asp
Ser Leu Ile Val Lys 130 135 140 Gly Phe Asn Val Val Ser Ala Trp Ala
Leu Gln Leu Gly Pro Lys Asp 145 150 155 160 Ala Ser Arg Gln Val Tyr
Ile Cys Ser Asn Asn Ile Gln Ala Arg Gln 165 170 175 Gln Val Ile Glu
Leu Ala Arg Gln Leu Asn Phe Ile Pro Ile Asp Leu 180 185 190 Gly Ser
Leu Ser Ser Ala Arg Glu Ile Glu Asn Leu Pro Leu Arg Leu 195 200 205
Phe Thr Leu Trp Arg Gly Pro Val Val Val Ala Ile Ser Leu Ala Thr 210
215 220 Phe Phe Phe Leu Tyr Ser Phe Val Arg Asp Val Ile His Pro Tyr
Ala 225 230 235 240 Arg Asn Gln Gln Ser Asp Phe Tyr Lys Ile Pro Ile
Glu Ile Val Asn 245 250 255 Lys Thr Leu Pro Ile Val Ala Ile Thr Leu
Leu Ser Leu Val Tyr Leu 260 265 270 Ala Gly Leu Leu Ala Ala Ala Tyr
Gln Leu Tyr Tyr Gly Thr Lys Tyr 275 280 285 Arg Arg Phe Pro Pro Trp
Leu Glu Thr Trp Leu Gln Cys Arg Lys Gln 290 295 300 Leu Gly Leu Leu
Ser Phe Phe Phe Ala Met Val His Val Ala Tyr Ser 305 310 315 320 Leu
Cys Leu Pro Met Arg Arg Ser Glu Arg Tyr Leu Phe Leu Asn Met 325 330
335 Ala Tyr Gln Gln Val His Ala Asn Ile Glu Asn Ser Trp Asn Glu Glu
340 345 350 Glu Val Trp Arg Ile Glu Met Tyr Ile Ser Phe Gly Ile Met
Ser Leu 355 360 365 Gly Leu Leu Ser Leu Leu Ala Val Thr Ser Ile Pro
Ser Val Ser Asn 370 375 380 Ala Leu Asn Trp Arg Glu Phe Ser Phe Ile
Gln Ser Thr Leu Gly Tyr 385 390 395 400 Val Ala Leu Leu Ile Ser Thr
Phe His Val Leu Ile Tyr Gly Trp Lys 405 410 415 Arg Ala Phe Glu Glu
Glu Tyr Tyr Arg Phe Tyr Thr Pro Pro Asn Phe 420 425 430 Val Leu Ala
Leu Val Leu Pro Ser Ile Val Ile Leu Gly Lys Ile Ile 435 440 445 Leu
Phe Leu Pro Cys Ile Ser Gln Lys Leu Lys Arg Ile Lys Lys Gly 450 455
460 Trp Glu Lys Ser Gln Phe Leu Glu Glu Gly Ile Gly Gly Thr Ile Pro
465 470 475 480 His Val Ser Pro Glu Arg Val Thr Val Met 485 490
<210> SEQ ID NO 12 <211> LENGTH: 1214 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: TrpM4 <400> SEQUENCE: 12 Met
Val Val Pro Glu Lys Glu Gln Ser Trp Ile Pro Lys Ile Phe Lys 1 5 10
15 Lys Lys Thr Cys Thr Thr Phe Ile Val Asp Ser Thr Asp Pro Gly Gly
20 25 30 Thr Leu Cys Gln Cys Gly Arg Pro Arg Thr Ala His Pro Ala
Val Ala 35 40 45 Met Glu Asp Ala Phe Gly Ala Ala Val Val Thr Val
Trp Asp Ser Asp 50 55 60 Ala His Thr Thr Glu Lys Pro Thr Asp Ala
Tyr Gly Glu Leu Asp Phe 65 70 75 80 Thr Gly Ala Gly Arg Lys His Ser
Asn Phe Leu Arg Leu Ser Asp Arg 85 90 95 Thr Asp Pro Ala Ala Val
Tyr Ser Leu Val Thr Arg Thr Trp Gly Phe 100 105 110 Arg Ala Pro Asn
Leu Val Val Ser Val Leu Gly Gly Ser Gly Gly Pro 115 120 125 Val Leu
Gln Thr Trp Leu Gln Asp Leu Leu Arg Arg Gly Leu Val Arg 130 135 140
Ala Ala Gln Ser Thr Gly Ala Trp Ile Val Thr Gly Gly Leu His Thr 145
150 155 160 Gly Ile Gly Arg His Val Gly Val Ala Val Arg Asp His Gln
Met Ala 165 170 175 Ser Thr Gly Gly Thr Lys Val Val Ala Met Gly Val
Ala Pro Trp Gly 180 185 190 Val Val Arg Asn Arg Asp Thr Leu Ile Asn
Pro Lys Gly Ser Phe Pro 195 200 205 Ala Arg Tyr Arg Trp Arg Gly Asp
Pro Glu Asp Gly Val Gln Phe Pro 210 215 220 Leu Asp Tyr Asn Tyr Ser
Ala Phe Phe Leu Val Asp Asp Gly Thr His 225 230 235 240 Gly Cys Leu
Gly Gly Glu Asn Arg Phe Arg Leu Arg Leu Glu Ser Tyr 245 250 255 Ile
Ser Gln Gln Lys Thr Gly Val Gly Gly Thr Gly Ile Asp Ile Pro 260 265
270 Val Leu Leu Leu Leu Ile Asp Gly Asp Glu Lys Met Leu Thr Arg Ile
275 280 285 Glu Asn Ala Thr Gln Ala Gln Leu Pro Cys Leu Leu Val Ala
Gly Ser 290 295 300 Gly Gly Ala Ala Asp Cys Leu Ala Glu Thr Leu Glu
Asp Thr Leu Ala 305 310 315 320 Pro Gly Ser Gly Gly Ala Arg Gln Gly
Glu Ala Arg Asp Arg Ile Arg 325 330 335 Arg Phe Phe Pro Lys Gly Asp
Leu Glu Val Leu Gln Ala Gln Val Glu 340 345 350 Arg Ile Met Thr Arg
Lys Glu Leu Leu Thr Val Tyr Ser Ser Glu Asp 355 360 365 Gly Ser Glu
Glu Phe Glu Thr Ile Val Leu Lys Ala Leu Val Lys Ala 370 375 380 Cys
Gly Ser Ser Glu Ala Ser Ala Tyr Leu Asp Glu Leu Arg Leu Ala 385 390
395 400 Val Ala Trp Asn Arg Val Asp Ile Ala Gln Ser Glu Leu Phe Arg
Gly 405 410 415 Asp Ile Gln Trp Arg Ser Phe His Leu Glu Ala Ser Leu
Met Asp Ala 420 425 430 Leu Leu Asn Asp Arg Pro Glu Phe Val Arg Leu
Leu Ile Ser His Gly 435 440 445 Leu Ser Leu Gly His Phe Leu Thr Pro
Met Arg Leu Ala Gln Leu Tyr 450 455 460
Ser Ala Ala Pro Ser Asn Ser Leu Ile Arg Asn Leu Leu Asp Gln Ala 465
470 475 480 Ser His Ser Ala Gly Thr Lys Ala Pro Ala Leu Lys Gly Gly
Ala Ala 485 490 495 Glu Leu Arg Pro Pro Asp Val Gly His Val Leu Arg
Met Leu Leu Gly 500 505 510 Lys Met Cys Ala Pro Arg Tyr Pro Ser Gly
Gly Ala Trp Asp Pro His 515 520 525 Pro Gly Gln Gly Phe Gly Glu Ser
Met Tyr Leu Leu Ser Asp Lys Ala 530 535 540 Thr Ser Pro Leu Ser Leu
Asp Ala Gly Leu Gly Gln Ala Pro Trp Ser 545 550 555 560 Asp Leu Leu
Leu Trp Ala Leu Leu Leu Asn Arg Ala Gln Met Ala Met 565 570 575 Tyr
Phe Trp Glu Met Gly Ser Asn Ala Val Ser Ser Ala Leu Gly Ala 580 585
590 Cys Leu Leu Leu Arg Val Met Ala Arg Leu Glu Pro Asp Ala Glu Glu
595 600 605 Ala Ala Arg Arg Lys Asp Leu Ala Phe Lys Phe Glu Gly Met
Gly Val 610 615 620 Asp Leu Phe Gly Glu Cys Tyr Arg Ser Ser Glu Val
Arg Ala Ala Arg 625 630 635 640 Leu Leu Leu Arg Arg Cys Pro Leu Trp
Gly Asp Ala Thr Cys Leu Gln 645 650 655 Leu Ala Met Gln Ala Asp Ala
Arg Ala Phe Phe Ala Gln Asp Gly Val 660 665 670 Gln Ser Leu Leu Thr
Gln Lys Trp Trp Gly Asp Met Ala Ser Thr Thr 675 680 685 Pro Ile Trp
Ala Leu Val Leu Ala Phe Phe Cys Pro Pro Leu Ile Tyr 690 695 700 Thr
Arg Leu Ile Thr Phe Arg Lys Ser Glu Glu Glu Pro Thr Arg Glu 705 710
715 720 Glu Leu Glu Phe Asp Met Asp Ser Val Ile Asn Gly Glu Gly Pro
Val 725 730 735 Gly Thr Ala Asp Pro Ala Glu Lys Thr Pro Leu Gly Val
Pro Arg Gln 740 745 750 Ser Gly Arg Pro Gly Cys Cys Gly Gly Arg Cys
Gly Gly Arg Arg Cys 755 760 765 Leu Arg Arg Trp Phe His Phe Trp Gly
Ala Pro Val Thr Ile Phe Met 770 775 780 Gly Asn Val Val Ser Tyr Leu
Leu Phe Leu Leu Leu Phe Ser Arg Val 785 790 795 800 Leu Leu Val Asp
Phe Gln Pro Ala Pro Pro Gly Ser Leu Glu Leu Leu 805 810 815 Leu Tyr
Phe Trp Ala Phe Thr Leu Leu Cys Glu Glu Leu Arg Gln Gly 820 825 830
Leu Ser Gly Gly Gly Gly Ser Leu Ala Ser Gly Gly Pro Gly Pro Gly 835
840 845 His Ala Ser Leu Ser Gln Arg Leu Arg Leu Tyr Leu Ala Asp Ser
Trp 850 855 860 Asn Gln Cys Asp Leu Val Ala Leu Thr Cys Phe Leu Leu
Gly Val Gly 865 870 875 880 Cys Arg Leu Thr Pro Gly Leu Tyr His Leu
Gly Arg Thr Val Leu Cys 885 890 895 Ile Asp Phe Met Val Phe Thr Val
Arg Leu Leu His Ile Phe Thr Val 900 905 910 Asn Lys Gln Leu Gly Pro
Lys Ile Val Ile Val Ser Lys Met Met Lys 915 920 925 Asp Val Phe Phe
Phe Leu Phe Phe Leu Gly Val Trp Leu Val Ala Tyr 930 935 940 Gly Val
Ala Thr Glu Gly Leu Leu Arg Pro Arg Asp Ser Asp Phe Pro 945 950 955
960 Ser Ile Leu Arg Arg Val Phe Tyr Arg Pro Tyr Leu Gln Ile Phe Gly
965 970 975 Gln Ile Pro Gln Glu Asp Met Asp Val Ala Leu Met Glu His
Ser Asn 980 985 990 Cys Ser Ser Glu Pro Gly Phe Trp Ala His Pro Pro
Gly Ala Gln Ala 995 1000 1005 Gly Thr Cys Val Ser Gln Tyr Ala Asn
Trp Leu Val Val Leu Leu Leu 1010 1015 1020 Val Ile Phe Leu Leu Val
Ala Asn Ile Leu Leu Val Asn Leu Leu Ile 1025 1030 1035 1040 Ala Met
Phe Ser Tyr Thr Phe Gly Lys Val Gln Gly Asn Ser Asp Leu 1045 1050
1055 Tyr Trp Lys Ala Gln Arg Tyr Arg Leu Ile Arg Glu Phe His Ser
Arg 1060 1065 1070 Pro Ala Leu Ala Pro Pro Phe Ile Val Ile Ser His
Leu Arg Leu Leu 1075 1080 1085 Leu Arg Gln Leu Cys Arg Arg Pro Arg
Ser Pro Gln Pro Ser Ser Pro 1090 1095 1100 Ala Leu Glu His Phe Arg
Val Tyr Leu Ser Lys Glu Ala Glu Arg Lys 1105 1110 1115 1120 Leu Leu
Thr Trp Glu Ser Val His Lys Glu Asn Phe Leu Leu Ala Arg 1125 1130
1135 Ala Arg Asp Lys Arg Glu Ser Asp Ser Glu Arg Leu Lys Arg Thr
Ser 1140 1145 1150 Gln Lys Val Asp Leu Ala Leu Lys Gln Leu Gly His
Ile Arg Glu Tyr 1155 1160 1165 Glu Gln Arg Leu Lys Val Leu Glu Arg
Glu Val Gln Gln Cys Ser Arg 1170 1175 1180 Val Leu Gly Trp Val Ala
Glu Ala Leu Ser Arg Ser Ala Leu Leu Pro 1185 1190 1195 1200 Pro Gly
Gly Pro Pro Pro Pro Asp Leu Pro Gly Ser Lys Asp 1205 1210
<210> SEQ ID NO 13 <211> LENGTH: 188 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: TDGF1 <400> SEQUENCE: 13 Met
Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp Ile 1 5 10
15 Met Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly Leu Gly
20 25 30 His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala Phe
Arg Asp 35 40 45 Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg
Pro Arg Ser Ser 50 55 60 Gln Arg Val Pro Pro Met Gly Ile Gln His
Ser Lys Glu Leu Asn Arg 65 70 75 80 Thr Cys Cys Leu Asn Gly Gly Thr
Cys Met Leu Gly Ser Phe Cys Ala 85 90 95 Cys Pro Pro Ser Phe Tyr
Gly Arg Asn Cys Glu His Asp Val Arg Lys 100 105 110 Glu Asn Cys Gly
Ser Val Pro His Asp Thr Trp Leu Pro Lys Lys Cys 115 120 125 Ser Leu
Cys Lys Cys Trp His Gly Gln Leu Arg Cys Phe Pro Gln Ala 130 135 140
Phe Leu Pro Gly Cys Asp Gly Leu Val Met Asp Glu His Leu Val Ala 145
150 155 160 Ser Arg Thr Pro Glu Leu Pro Pro Ser Ala Arg Thr Thr Thr
Phe Met 165 170 175 Leu Val Gly Ile Cys Leu Ser Ile Gln Ser Tyr Tyr
180 185 <210> SEQ ID NO 14 <211> LENGTH: 1033
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CD21
<400> SEQUENCE: 14 Met Gly Ala Ala Gly Leu Leu Gly Val Phe
Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly Ile Ser Cys Gly
Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser
Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45 Tyr Ser Cys Ser
Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60 Cys Ile
Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro 65 70 75 80
Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85
90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly
Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn
Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly
Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val Ser Val Phe Pro Leu Glu
Cys Pro Ala Leu Pro Met Ile 145 150 155 160 His Asn Gly His His Thr
Ser Glu Asn Val Gly Ser Ile Ala Pro Gly 165 170 175 Leu Ser Val Thr
Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu 180 185 190 Lys Ile
Ile Asn Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro 195 200 205
Thr Cys Glu Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly 210
215 220 Lys Val Lys Glu Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn
Phe 225 230 235 240 Phe Cys Asp Glu Gly Tyr Arg Leu Gln Gly Pro Pro
Ser Ser Arg Cys 245 250 255 Val Ile Ala Gly Gln Gly Val Ala Trp Thr
Lys Met Pro Val Cys Glu 260 265 270 Glu Ile Phe Cys Pro Ser Pro Pro
Pro Ile Leu Asn Gly Arg His Ile 275 280 285 Gly Asn Ser Leu Ala Asn
Val Ser Tyr Gly Ser Ile Val Thr Tyr Thr 290 295 300 Cys Asp Pro Asp
Pro Glu Glu Gly Val Asn Phe Ile Leu Ile Gly Glu 305 310 315 320 Ser
Thr Leu Arg Cys Thr Val Asp Ser Gln Lys Thr Gly Thr Trp Ser 325 330
335 Gly Pro Ala Pro Arg Cys Glu Leu Ser Thr Ser Ala Val Gln Cys Pro
340 345 350 His Pro Gln Ile Leu Arg Gly Arg Met Val Ser Gly Gln Lys
Asp Arg 355 360 365 Tyr Thr Tyr Asn Asp Thr Val Ile Phe Ala Cys Met
Phe Gly Phe Thr 370 375 380 Leu Lys Gly Ser Lys Gln Ile Arg Cys Asn
Ala Gln Gly Thr Trp Glu 385 390 395 400 Pro Ser Ala Pro Val Cys Glu
Lys Glu Cys Gln Ala Pro Pro Asn Ile 405 410 415 Leu Asn Gly Gln Lys
Glu Asp Arg His Met Val Arg Phe Asp Pro Gly 420 425 430 Thr Ser Ile
Lys Tyr Ser Cys Asn Pro Gly Tyr Val Leu Val Gly Glu 435 440 445 Glu
Ser Ile Gln Cys Thr Ser Glu Gly Val Trp Thr Pro Pro Val Pro 450 455
460 Gln Cys Lys Val Ala Ala Cys Glu Ala Thr Gly Arg Gln Leu Leu Thr
465 470 475 480 Lys Pro Gln His Gln Phe Val Arg Pro Asp Val Asn Ser
Ser Cys Gly
485 490 495 Glu Gly Tyr Lys Leu Ser Gly Ser Val Tyr Gln Glu Cys Gln
Gly Thr 500 505 510 Ile Pro Trp Phe Met Glu Ile Arg Leu Cys Lys Glu
Ile Thr Cys Pro 515 520 525 Pro Pro Pro Val Ile Tyr Asn Gly Ala His
Thr Gly Ser Ser Leu Glu 530 535 540 Asp Phe Pro Tyr Gly Thr Thr Val
Thr Tyr Thr Cys Asn Pro Gly Pro 545 550 555 560 Glu Arg Gly Val Glu
Phe Ser Leu Ile Gly Glu Ser Thr Ile Arg Cys 565 570 575 Thr Ser Asn
Asp Gln Glu Arg Gly Thr Trp Ser Gly Pro Ala Pro Leu 580 585 590 Cys
Lys Leu Ser Leu Leu Ala Val Gln Cys Ser His Val His Ile Ala 595 600
605 Asn Gly Tyr Lys Ile Ser Gly Lys Glu Ala Pro Tyr Phe Tyr Asn Asp
610 615 620 Thr Val Thr Phe Lys Cys Tyr Ser Gly Phe Thr Leu Lys Gly
Ser Ser 625 630 635 640 Gln Ile Arg Cys Lys Ala Asp Asn Thr Trp Asp
Pro Glu Ile Pro Val 645 650 655 Cys Glu Lys Glu Thr Cys Gln His Val
Arg Gln Ser Leu Gln Glu Leu 660 665 670 Pro Ala Gly Ser Arg Val Glu
Leu Val Asn Thr Ser Cys Gln Asp Gly 675 680 685 Tyr Gln Leu Thr Gly
His Ala Tyr Gln Met Cys Gln Asp Ala Glu Asn 690 695 700 Gly Ile Trp
Phe Lys Lys Ile Pro Leu Cys Lys Val Ile His Cys His 705 710 715 720
Pro Pro Pro Val Ile Val Asn Gly Lys His Thr Gly Met Met Ala Glu 725
730 735 Asn Phe Leu Tyr Gly Asn Glu Val Ser Tyr Glu Cys Asp Gln Gly
Phe 740 745 750 Tyr Leu Leu Gly Glu Lys Lys Leu Gln Cys Arg Ser Asp
Ser Lys Gly 755 760 765 His Gly Ser Trp Ser Gly Pro Ser Pro Gln Cys
Leu Arg Ser Pro Pro 770 775 780 Val Thr Arg Cys Pro Asn Pro Glu Val
Lys His Gly Tyr Lys Leu Asn 785 790 795 800 Lys Thr His Ser Ala Tyr
Ser His Asn Asp Ile Val Tyr Val Asp Cys 805 810 815 Asn Pro Gly Phe
Ile Met Asn Gly Ser Arg Val Ile Arg Cys His Thr 820 825 830 Asp Asn
Thr Trp Val Pro Gly Val Pro Thr Cys Ile Lys Lys Ala Phe 835 840 845
Ile Gly Cys Pro Pro Pro Pro Lys Thr Pro Asn Gly Asn His Thr Gly 850
855 860 Gly Asn Ile Ala Arg Phe Ser Pro Gly Met Ser Ile Leu Tyr Ser
Cys 865 870 875 880 Asp Gln Gly Tyr Leu Leu Val Gly Glu Ala Leu Leu
Leu Cys Thr His 885 890 895 Glu Gly Thr Trp Ser Gln Pro Ala Pro His
Cys Lys Glu Val Asn Cys 900 905 910 Ser Ser Pro Ala Asp Met Asp Gly
Ile Gln Lys Gly Leu Glu Pro Arg 915 920 925 Lys Met Tyr Gln Tyr Gly
Ala Val Val Thr Leu Glu Cys Glu Asp Gly 930 935 940 Tyr Met Leu Glu
Gly Ser Pro Gln Ser Gln Cys Gln Ser Asp His Gln 945 950 955 960 Trp
Asn Pro Pro Leu Ala Val Cys Arg Ser Arg Ser Leu Ala Pro Val 965 970
975 Leu Cys Gly Ile Ala Ala Gly Leu Ile Leu Leu Thr Phe Leu Ile Val
980 985 990 Ile Thr Leu Tyr Val Ile Ser Lys His Arg Glu Arg Asn Tyr
Tyr Thr 995 1000 1005 Asp Thr Ser Gln Lys Glu Ala Phe His Leu Glu
Ala Arg Glu Val Tyr 1010 1015 1020 Ser Val Asp Pro Tyr Asn Pro Ala
Ser 1025 1030 <210> SEQ ID NO 15 <211> LENGTH: 229
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CD79B
<400> SEQUENCE: 15 Met Ala Arg Leu Ala Leu Ser Pro Val Pro
Ser His Trp Met Val Ala 1 5 10 15 Leu Leu Leu Leu Leu Ser Ala Glu
Pro Val Pro Ala Ala Arg Ser Glu 20 25 30 Asp Arg Tyr Arg Asn Pro
Lys Gly Ser Ala Cys Ser Arg Ile Trp Gln 35 40 45 Ser Pro Arg Phe
Ile Ala Arg Lys Arg Gly Phe Thr Val Lys Met His 50 55 60 Cys Tyr
Met Asn Ser Ala Ser Gly Asn Val Ser Trp Leu Trp Lys Gln 65 70 75 80
Glu Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu Lys Gly Arg Met 85
90 95 Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr Ile Gln Gly
Ile 100 105 110 Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln Gln Lys
Cys Asn Asn 115 120 125 Thr Ser Glu Val Tyr Gln Gly Cys Gly Thr Glu
Leu Arg Val Met Gly 130 135 140 Phe Ser Thr Leu Ala Gln Leu Lys Gln
Arg Asn Thr Leu Lys Asp Gly 145 150 155 160 Ile Ile Met Ile Gln Thr
Leu Leu Ile Ile Leu Phe Ile Ile Val Pro 165 170 175 Ile Phe Leu Leu
Leu Asp Lys Asp Asp Ser Lys Ala Gly Met Glu Glu 180 185 190 Asp His
Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu 195 200 205
Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu 210
215 220 His Pro Gly Gln Glu 225 <210> SEQ ID NO 16
<211> LENGTH: 508 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
FcRH2 <400> SEQUENCE: 16 Met Leu Leu Trp Ser Leu Leu Val Ile
Phe Asp Ala Val Thr Glu Gln 1 5 10 15 Ala Asp Ser Leu Thr Leu Val
Ala Pro Ser Ser Val Phe Glu Gly Asp 20 25 30 Ser Ile Val Leu Lys
Cys Gln Gly Glu Gln Asn Trp Lys Ile Gln Lys 35 40 45 Met Ala Tyr
His Lys Asp Asn Lys Glu Leu Ser Val Phe Lys Lys Phe 50 55 60 Ser
Asp Phe Leu Ile Gln Ser Ala Val Leu Ser Asp Ser Gly Asn Tyr 65 70
75 80 Phe Cys Ser Thr Lys Gly Gln Leu Phe Leu Trp Asp Lys Thr Ser
Asn 85 90 95 Ile Val Lys Ile Lys Val Gln Glu Leu Phe Gln Arg Pro
Val Leu Thr 100 105 110 Ala Ser Ser Phe Gln Pro Ile Glu Gly Gly Pro
Val Ser Leu Lys Cys 115 120 125 Glu Thr Arg Leu Ser Pro Gln Arg Leu
Asp Val Gln Leu Gln Phe Cys 130 135 140 Phe Phe Arg Glu Asn Gln Val
Leu Gly Ser Gly Trp Ser Ser Ser Pro 145 150 155 160 Glu Leu Gln Ile
Ser Ala Val Trp Ser Glu Asp Thr Gly Ser Tyr Trp 165 170 175 Cys Lys
Ala Glu Thr Val Thr His Arg Ile Arg Lys Gln Ser Leu Gln 180 185 190
Ser Gln Ile His Val Gln Arg Ile Pro Ile Ser Asn Val Ser Leu Glu 195
200 205 Ile Arg Ala Pro Gly Gly Gln Val Thr Glu Gly Gln Lys Leu Ile
Leu 210 215 220 Leu Cys Ser Val Ala Gly Gly Thr Gly Asn Val Thr Phe
Ser Trp Tyr 225 230 235 240 Arg Glu Ala Thr Gly Thr Ser Met Gly Lys
Lys Thr Gln Arg Ser Leu 245 250 255 Ser Ala Glu Leu Glu Ile Pro Ala
Val Lys Glu Ser Asp Ala Gly Lys 260 265 270 Tyr Tyr Cys Arg Ala Asp
Asn Gly His Val Pro Ile Gln Ser Lys Val 275 280 285 Val Asn Ile Pro
Val Arg Ile Pro Val Ser Arg Pro Val Leu Thr Leu 290 295 300 Arg Ser
Pro Gly Ala Gln Ala Ala Val Gly Asp Leu Leu Glu Leu His 305 310 315
320 Cys Glu Ala Leu Arg Gly Ser Pro Pro Ile Leu Tyr Gln Phe Tyr His
325 330 335 Glu Asp Val Thr Leu Gly Asn Ser Ser Ala Pro Ser Gly Gly
Gly Ala 340 345 350 Ser Phe Asn Leu Ser Leu Thr Ala Glu His Ser Gly
Asn Tyr Ser Cys 355 360 365 Glu Ala Asn Asn Gly Leu Gly Ala Gln Cys
Ser Glu Ala Val Pro Val 370 375 380 Ser Ile Ser Gly Pro Asp Gly Tyr
Arg Arg Asp Leu Met Thr Ala Gly 385 390 395 400 Val Leu Trp Gly Leu
Phe Gly Val Leu Gly Phe Thr Gly Val Ala Leu 405 410 415 Leu Leu Tyr
Ala Leu Phe His Lys Ile Ser Gly Glu Ser Ser Ala Thr 420 425 430 Asn
Glu Pro Arg Gly Ala Ser Arg Pro Asn Pro Gln Glu Phe Thr Tyr 435 440
445 Ser Ser Pro Thr Pro Asp Met Glu Glu Leu Gln Pro Val Tyr Val Asn
450 455 460 Val Gly Ser Val Asp Val Asp Val Val Tyr Ser Gln Val Trp
Ser Met 465 470 475 480 Gln Gln Pro Glu Ser Ser Ala Asn Ile Arg Thr
Leu Leu Glu Asn Lys 485 490 495 Asp Ser Gln Val Ile Tyr Ser Ser Val
Lys Lys Ser 500 505
<210> SEQ ID NO 17 <211> LENGTH: 1255 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: HER2 <400> SEQUENCE: 17 Met
Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu 1 5 10
15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys
20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu
Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu
Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu
Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala His
Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val
Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala Val
Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val Thr
Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140
Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 145
150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His
Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg
Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met Cys Lys Gly Ser
Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys Gln Ser Leu Thr
Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg Cys Lys Gly Pro
Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235 240 Ala Ala Gly
Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255 His
Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265
270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn
Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro
Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr Gln
Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys Tyr
Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val Thr
Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile Phe
Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380 Pro
Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385 390
395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp
Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln
Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu
Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu Gly Leu Arg Ser
Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu Ile His His Asn
Thr His Leu Cys Phe Val His Thr Val 465 470 475 480 Pro Trp Asp Gln
Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495 Ala Asn
Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510
Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515
520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu
Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala
Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln Pro Gln
Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln Cys
Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val Ala
Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met Pro
Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro Cys
Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630 635
640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser
645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val
Phe Gly 660 665 670 Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys
Tyr Thr Met Arg 675 680 685 Arg Leu Leu Gln Glu Thr Glu Leu Val Glu
Pro Leu Thr Pro Ser Gly 690 695 700 Ala Met Pro Asn Gln Ala Gln Met
Arg Ile Leu Lys Glu Thr Glu Leu 705 710 715 720 Arg Lys Val Lys Val
Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735 Gly Ile Trp
Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750 Lys
Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760
765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg
770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr
Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg
Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser Gln Asp Leu Leu Asn Trp
Cys Met Gln Ile Ala Lys Gly 820 825 830 Met Ser Tyr Leu Glu Asp Val
Arg Leu Val His Arg Asp Leu Ala Ala 835 840 845 Arg Asn Val Leu Val
Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860 Gly Leu Ala
Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 865 870 875 880
Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg 885
890 895 Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr
Val 900 905 910 Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly
Ile Pro Ala 915 920 925 Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu
Arg Leu Pro Gln Pro 930 935 940 Pro Ile Cys Thr Ile Asp Val Tyr Met
Ile Met Val Lys Cys Trp Met 945 950 955 960 Ile Asp Ser Glu Cys Arg
Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965 970 975 Ser Arg Met Ala
Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 980 985 990 Asp Leu
Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 995 1000
1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr
Leu 1010 1015 1020 Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala
Pro Gly Ala Gly 1025 1030 1035 1040 Gly Met Val His His Arg His Arg
Ser Ser Ser Thr Arg Ser Gly Gly 1045 1050 1055 Gly Asp Leu Thr Leu
Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg 1060 1065 1070 Ser Pro
Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly 1075 1080
1085 Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr
His 1090 1095 1100 Asp Pro Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro
Thr Val Pro Leu 1105 1110 1115 1120 Pro Ser Glu Thr Asp Gly Tyr Val
Ala Pro Leu Thr Cys Ser Pro Gln 1125 1130 1135 Pro Glu Tyr Val Asn
Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro 1140 1145 1150 Arg Glu
Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu 1155 1160
1165 Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp
Val 1170 1175 1180 Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr
Leu Thr Pro Gln 1185 1190 1195 1200 Gly Gly Ala Ala Pro Gln Pro His
Pro Pro Pro Ala Phe Ser Pro Ala 1205 1210 1215 Phe Asp Asn Leu Tyr
Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala 1220 1225 1230 Pro Pro
Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1235 1240
1245 Leu Gly Leu Asp Val Pro Val 1250 1255 <210> SEQ ID NO 18
<211> LENGTH: 344
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CEACAM6
<400> SEQUENCE: 18 Met Gly Pro Pro Ser Ala Pro Pro Cys Arg
Leu His Val Pro Trp Lys 1 5 10 15 Glu Val Leu Leu Thr Ala Ser Leu
Leu Thr Phe Trp Asn Pro Pro Thr 20 25 30 Thr Ala Lys Leu Thr Ile
Glu Ser Thr Pro Phe Asn Val Ala Glu Gly 35 40 45 Lys Glu Val Leu
Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly 50 55 60 Tyr Ser
Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val 65 70 75 80
Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser 85
90 95 Gly Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn
Val 100 105 110 Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile
Lys Ser Asp 115 120 125 Leu Val Asn Glu Glu Ala Thr Gly Gln Phe His
Val Tyr Pro Glu Leu 130 135 140 Pro Lys Pro Ser Ile Ser Ser Asn Asn
Ser Asn Pro Val Glu Asp Lys 145 150 155 160 Asp Ala Val Ala Phe Thr
Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr 165 170 175 Leu Trp Trp Val
Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 180 185 190 Leu Ser
Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn 195 200 205
Asp Ala Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn 210
215 220 Arg Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Val
Pro 225 230 235 240 Thr Ile Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly
Glu Asn Leu Asn 245 250 255 Leu Ser Cys His Ala Ala Ser Asn Pro Pro
Ala Gln Tyr Ser Trp Phe 260 265 270 Ile Asn Gly Thr Phe Gln Gln Ser
Thr Gln Glu Leu Phe Ile Pro Asn 275 280 285 Ile Thr Val Asn Asn Ser
Gly Ser Tyr Met Cys Gln Ala His Asn Ser 290 295 300 Ala Thr Gly Leu
Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly 305 310 315 320 Ser
Ala Pro Val Leu Ser Ala Val Ala Thr Val Gly Ile Thr Ile Gly 325 330
335 Val Leu Ala Arg Val Ala Leu Ile 340 <210> SEQ ID NO 19
<211> LENGTH: 411 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
DPEP1 <400> SEQUENCE: 19 Met Trp Ser Gly Trp Trp Leu Trp Pro
Leu Val Ala Val Cys Thr Ala 1 5 10 15 Asp Phe Phe Arg Asp Glu Ala
Glu Arg Ile Met Arg Asp Ser Pro Val 20 25 30 Ile Asp Gly His Asn
Asp Leu Pro Trp Gln Leu Leu Asp Met Phe Asn 35 40 45 Asn Arg Leu
Gln Asp Glu Arg Ala Asn Leu Thr Thr Leu Ala Gly Thr 50 55 60 His
Thr Asn Ile Pro Lys Leu Arg Ala Gly Phe Val Gly Gly Gln Phe 65 70
75 80 Trp Ser Val Tyr Thr Pro Cys Asp Thr Gln Asn Lys Asp Ala Val
Arg 85 90 95 Arg Thr Leu Glu Gln Met Asp Val Val His Arg Met Cys
Arg Met Tyr 100 105 110 Pro Glu Thr Phe Leu Tyr Val Thr Ser Ser Ala
Gly Ile Arg Gln Ala 115 120 125 Phe Arg Glu Gly Lys Val Ala Ser Leu
Ile Gly Val Glu Gly Gly His 130 135 140 Ser Ile Asp Ser Ser Leu Gly
Val Leu Arg Ala Leu Tyr Gln Leu Gly 145 150 155 160 Met Arg Tyr Leu
Thr Leu Thr His Ser Cys Asn Thr Pro Trp Ala Asp 165 170 175 Asn Trp
Leu Val Asp Thr Gly Asp Ser Glu Pro Gln Ser Gln Gly Leu 180 185 190
Ser Pro Phe Gly Gln Arg Val Val Lys Glu Leu Asn Arg Leu Gly Val 195
200 205 Leu Ile Asp Leu Ala His Val Ser Val Ala Thr Met Lys Ala Thr
Leu 210 215 220 Gln Leu Ser Arg Ala Pro Val Ile Phe Ser His Ser Ser
Ala Tyr Ser 225 230 235 240 Val Cys Ala Ser Arg Arg Asn Val Pro Asp
Asp Val Leu Arg Leu Val 245 250 255 Lys Gln Thr Asp Ser Leu Val Met
Val Asn Phe Tyr Asn Asn Tyr Ile 260 265 270 Ser Cys Thr Asn Lys Ala
Asn Leu Ser Gln Val Ala Asp His Leu Asp 275 280 285 His Ile Lys Glu
Val Ala Gly Ala Arg Ala Val Gly Phe Gly Gly Asp 290 295 300 Phe Asp
Gly Val Pro Arg Val Pro Glu Gly Leu Glu Asp Val Ser Lys 305 310 315
320 Tyr Pro Asp Leu Ile Ala Glu Leu Leu Arg Arg Asn Trp Thr Glu Ala
325 330 335 Glu Val Lys Gly Ala Leu Ala Asp Asn Leu Leu Arg Val Phe
Glu Ala 340 345 350 Val Glu Gln Ala Ser Asn Leu Thr Gln Ala Pro Glu
Glu Glu Pro Ile 355 360 365 Pro Leu Asp Gln Leu Gly Gly Ser Cys Arg
Thr His Tyr Gly Tyr Ser 370 375 380 Ser Gly Ala Ser Ser Leu His Arg
His Trp Gly Leu Leu Leu Ala Ser 385 390 395 400 Leu Ala Pro Leu Val
Leu Cys Leu Ser Leu Leu 405 410 <210> SEQ ID NO 20
<211> LENGTH: 553 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
IL20Ra <400> SEQUENCE: 20 Met Arg Ala Pro Gly Arg Pro Ala Leu
Arg Pro Leu Pro Leu Pro Pro 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala
Ala Pro Trp Gly Arg Ala Val Pro Cys 20 25 30 Val Ser Gly Gly Leu
Pro Lys Pro Ala Asn Ile Thr Phe Leu Ser Ile 35 40 45 Asn Met Lys
Asn Val Leu Gln Trp Thr Pro Pro Glu Gly Leu Gln Gly 50 55 60 Val
Lys Val Thr Tyr Thr Val Gln Tyr Phe Ile Tyr Gly Gln Lys Lys 65 70
75 80 Trp Leu Asn Lys Ser Glu Cys Arg Asn Ile Asn Arg Thr Tyr Cys
Asp 85 90 95 Leu Ser Ala Glu Thr Ser Asp Tyr Glu His Gln Tyr Tyr
Ala Lys Val 100 105 110 Lys Ala Ile Trp Gly Thr Lys Cys Ser Lys Trp
Ala Glu Ser Gly Arg 115 120 125 Phe Tyr Pro Phe Leu Glu Thr Gln Ile
Gly Pro Pro Glu Val Ala Leu 130 135 140 Thr Thr Asp Glu Lys Ser Ile
Ser Val Val Leu Thr Ala Pro Glu Lys 145 150 155 160 Trp Lys Arg Asn
Pro Glu Asp Leu Pro Val Ser Met Gln Gln Ile Tyr 165 170 175 Ser Asn
Leu Lys Tyr Asn Val Ser Val Leu Asn Thr Lys Ser Asn Arg 180 185 190
Thr Trp Ser Gln Cys Val Thr Asn His Thr Leu Val Leu Thr Trp Leu 195
200 205 Glu Pro Asn Thr Leu Tyr Cys Val His Val Glu Ser Phe Val Pro
Gly 210 215 220 Pro Pro Arg Arg Ala Gln Pro Ser Glu Lys Gln Cys Ala
Arg Thr Leu 225 230 235 240 Lys Asp Gln Ser Ser Glu Phe Lys Ala Lys
Ile Ile Phe Trp Tyr Val 245 250 255 Leu Pro Ile Ser Ile Thr Val Phe
Leu Phe Ser Val Met Gly Tyr Ser 260 265 270 Ile Tyr Arg Tyr Ile His
Val Gly Lys Glu Lys His Pro Ala Asn Leu 275 280 285 Ile Leu Ile Tyr
Gly Asn Glu Phe Asp Lys Arg Phe Phe Val Pro Ala 290 295 300 Glu Lys
Ile Val Ile Asn Phe Ile Thr Leu Asn Ile Ser Asp Asp Ser 305 310 315
320 Lys Ile Ser His Gln Asp Met Ser Leu Leu Gly Lys Ser Ser Asp Val
325 330 335 Ser Ser Leu Asn Asp Pro Gln Pro Ser Gly Asn Leu Arg Pro
Pro Gln 340 345 350 Glu Glu Glu Glu Val Lys His Leu Gly Tyr Ala Ser
His Leu Met Glu 355 360 365 Ile Phe Cys Asp Ser Glu Glu Asn Thr Glu
Gly Thr Ser Phe Thr Gln 370 375 380 Gln Glu Ser Leu Ser Arg Thr Ile
Pro Pro Asp Lys Thr Val Ile Glu 385 390 395 400 Tyr Glu Tyr Asp Val
Arg Thr Thr Asp Ile Cys Ala Gly Pro Glu Glu 405 410 415 Gln Glu Leu
Ser Leu Gln Glu Glu Val Ser Thr Gln Gly Thr Leu Leu 420 425 430
Glu Ser Gln Ala Ala Leu Ala Val Leu Gly Pro Gln Thr Leu Gln Tyr 435
440 445 Ser Tyr Thr Pro Gln Leu Gln Asp Leu Asp Pro Leu Ala Gln Glu
His 450 455 460 Thr Asp Ser Glu Glu Gly Pro Glu Glu Glu Pro Ser Thr
Thr Leu Val 465 470 475 480 Asp Trp Asp Pro Gln Thr Gly Arg Leu Cys
Ile Pro Ser Leu Ser Ser 485 490 495 Phe Asp Gln Asp Ser Glu Gly Cys
Glu Pro Ser Glu Gly Asp Gly Leu 500 505 510 Gly Glu Glu Gly Leu Leu
Ser Arg Leu Tyr Glu Glu Pro Ala Pro Asp 515 520 525 Arg Pro Pro Gly
Glu Asn Glu Thr Tyr Leu Met Gln Phe Met Glu Glu 530 535 540 Trp Gly
Leu Tyr Val Gln Met Glu Asn 545 550 <210> SEQ ID NO 21
<211> LENGTH: 911 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
BCAN <400> SEQUENCE: 21 Met Ala Gln Leu Phe Leu Pro Leu Leu
Ala Ala Leu Val Leu Ala Gln 1 5 10 15 Ala Pro Ala Ala Leu Ala Asp
Val Leu Glu Gly Asp Ser Ser Glu Asp 20 25 30 Arg Ala Phe Arg Val
Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val 35 40 45 Leu Gly Gly
Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro 50 55 60 Pro
Pro Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr 65 70
75 80 Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly
Val 85 90 95 Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala
Leu Pro Ala 100 105 110 Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala
Leu Ser Glu Leu Arg 115 120 125 Pro Asn Asp Ser Gly Ile Tyr Arg Cys
Glu Val Gln His Gly Ile Asp 130 135 140 Asp Ser Ser Asp Ala Val Glu
Val Lys Val Lys Gly Val Val Phe Leu 145 150 155 160 Tyr Arg Glu Gly
Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln 165 170 175 Glu Ala
Cys Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu 180 185 190
Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu 195
200 205 Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala
Cys 210 215 220 Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr
Gly Val Val 225 230 235 240 Asp Pro Asp Asp Leu Tyr Asp Val Tyr Cys
Tyr Ala Glu Asp Leu Asn 245 250 255 Gly Glu Leu Phe Leu Gly Asp Pro
Pro Glu Lys Leu Thr Leu Glu Glu 260 265 270 Ala Arg Ala Tyr Cys Gln
Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly 275 280 285 Gln Leu Tyr Ala
Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly 290 295 300 Trp Leu
Ala Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln 305 310 315
320 Arg Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro
325 330 335 Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val
Tyr Cys 340 345 350 Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu
Ala Ser Asn Pro 355 360 365 Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu
Ala Ile Val Thr Val Thr 370 375 380 Glu Thr Leu Glu Glu Leu Gln Leu
Pro Gln Glu Ala Thr Glu Ser Glu 385 390 395 400 Ser Arg Gly Ala Ile
Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly Gly 405 410 415 Gly Ser Ser
Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu 420 425 430 Glu
Phe Glu Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu 435 440
445 Glu Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu
450 455 460 Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala
Leu Trp 465 470 475 480 Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro
Glu Ala Ser Leu Pro 485 490 495 Thr Glu Pro Ala Ala Gln Glu Lys Ser
Leu Ser Gln Ala Pro Ala Arg 500 505 510 Ala Val Leu Gln Pro Gly Ala
Ser Pro Leu Pro Asp Gly Glu Ser Glu 515 520 525 Ala Ser Arg Pro Pro
Arg Val His Gly Pro Pro Thr Glu Thr Leu Pro 530 535 540 Thr Pro Arg
Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val 545 550 555 560
Glu Ala Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly 565
570 575 Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala
Pro 580 585 590 Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg
Glu Leu Glu 595 600 605 Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala
Pro Ala Gly Thr Ser 610 615 620 Val Gln Ala Gln Pro Val Leu Pro Thr
Asp Ser Ala Ser Arg Gly Gly 625 630 635 640 Val Ala Val Val Pro Ala
Ser Gly Asp Cys Val Pro Ser Pro Cys His 645 650 655 Asn Gly Gly Thr
Cys Leu Glu Glu Glu Glu Gly Val Arg Cys Leu Cys 660 665 670 Leu Pro
Gly Tyr Gly Gly Asp Leu Cys Asp Val Gly Leu Arg Phe Cys 675 680 685
Asn Pro Gly Trp Asp Ala Phe Gln Gly Ala Cys Tyr Lys His Phe Ser 690
695 700 Thr Arg Arg Ser Trp Glu Glu Ala Glu Thr Gln Cys Arg Met Tyr
Gly 705 710 715 720 Ala His Leu Ala Ser Ile Ser Thr Pro Glu Glu Gln
Asp Phe Ile Asn 725 730 735 Asn Arg Tyr Arg Glu Tyr Gln Trp Ile Gly
Leu Asn Asp Arg Thr Ile 740 745 750 Glu Gly Asp Phe Leu Trp Ser Asp
Gly Val Pro Leu Leu Tyr Glu Asn 755 760 765 Trp Asn Pro Gly Gln Pro
Asp Ser Tyr Phe Leu Ser Gly Glu Asn Cys 770 775 780 Val Val Met Val
Trp His Asp Gln Gly Gln Trp Ser Asp Val Pro Cys 785 790 795 800 Asn
Tyr His Leu Ser Tyr Thr Cys Lys Met Gly Leu Val Ser Cys Gly 805 810
815 Pro Pro Pro Glu Leu Pro Leu Ala Gln Val Phe Gly Arg Pro Arg Leu
820 825 830 Arg Tyr Glu Val Asp Thr Val Leu Arg Tyr Arg Cys Arg Glu
Gly Leu 835 840 845 Ala Gln Arg Asn Leu Pro Leu Ile Arg Cys Gln Glu
Asn Gly Arg Trp 850 855 860 Glu Ala Pro Gln Ile Ser Cys Val Pro Arg
Arg Pro Ala Arg Ala Leu 865 870 875 880 His Pro Glu Glu Asp Pro Glu
Gly Arg Gln Gly Arg Leu Leu Gly Arg 885 890 895 Trp Lys Ala Leu Leu
Ile Pro Pro Ser Ser Pro Met Pro Gly Pro 900 905 910 <210> SEQ
ID NO 22 <211> LENGTH: 987 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: EphB2R <400> SEQUENCE: 22 Met Ala Leu Arg Arg
Leu Gly Ala Ala Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Ala Ala Val
Glu Glu Thr Leu Met Asp Ser Thr Thr Ala Thr Ala Glu 20 25 30 Leu
Gly Trp Met Val His Pro Pro Ser Gly Trp Glu Glu Val Ser Gly 35 40
45 Tyr Asp Glu Asn Met Asn Thr Ile Arg Thr Tyr Gln Val Cys Asn Val
50 55 60 Phe Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Lys Phe Ile
Arg Arg 65 70 75 80 Arg Gly Ala His Arg Ile His Val Glu Met Lys Phe
Ser Val Arg Asp 85 90 95 Cys Ser Ser Ile Pro Ser Val Pro Gly Ser
Cys Lys Glu Thr Phe Asn 100 105 110 Leu Tyr Tyr Tyr Glu Ala Asp Phe
Asp Ser Ala Thr Lys Thr Phe Pro 115 120 125 Asn Trp Met Glu Asn Pro
Trp Val Lys Val Asp Thr Ile Ala Ala Asp 130 135 140 Glu Ser Phe Ser
Gln Val Asp Leu Gly Gly Arg Val Met Lys Ile Asn 145 150 155 160 Thr
Glu Val Arg Ser Phe Gly Pro Val Ser Arg Ser Gly Phe Tyr Leu 165 170
175 Ala Phe Gln Asp Tyr Gly Gly Cys Met Ser Leu Ile Ala Val Arg Val
180 185 190
Phe Tyr Arg Lys Cys Pro Arg Ile Ile Gln Asn Gly Ala Ile Phe Gln 195
200 205 Glu Thr Leu Ser Gly Ala Glu Ser Thr Ser Leu Val Ala Ala Arg
Gly 210 215 220 Ser Cys Ile Ala Asn Ala Glu Glu Val Asp Val Pro Ile
Lys Leu Tyr 225 230 235 240 Cys Asn Gly Asp Gly Glu Trp Leu Val Pro
Ile Gly Arg Cys Met Cys 245 250 255 Lys Ala Gly Phe Glu Ala Val Glu
Asn Gly Thr Val Cys Arg Gly Cys 260 265 270 Pro Ser Gly Thr Phe Lys
Ala Asn Gln Gly Asp Glu Ala Cys Thr His 275 280 285 Cys Pro Ile Asn
Ser Arg Thr Thr Ser Glu Gly Ala Thr Asn Cys Val 290 295 300 Cys Arg
Asn Gly Tyr Tyr Arg Ala Asp Leu Asp Pro Leu Asp Met Pro 305 310 315
320 Cys Thr Thr Ile Pro Ser Ala Pro Gln Ala Val Ile Ser Ser Val Asn
325 330 335 Glu Thr Ser Leu Met Leu Glu Trp Thr Pro Pro Arg Asp Ser
Gly Gly 340 345 350 Arg Glu Asp Leu Val Tyr Asn Ile Ile Cys Lys Ser
Cys Gly Ser Gly 355 360 365 Arg Gly Ala Cys Thr Arg Cys Gly Asp Asn
Val Gln Tyr Ala Pro Arg 370 375 380 Gln Leu Gly Leu Thr Glu Pro Arg
Ile Tyr Ile Ser Asp Leu Leu Ala 385 390 395 400 His Thr Gln Tyr Thr
Phe Glu Ile Gln Ala Val Asn Gly Val Thr Asp 405 410 415 Gln Ser Pro
Phe Ser Pro Gln Phe Ala Ser Val Asn Ile Thr Thr Asn 420 425 430 Gln
Ala Ala Pro Ser Ala Val Ser Ile Met His Gln Val Ser Arg Thr 435 440
445 Val Asp Ser Ile Thr Leu Ser Trp Ser Gln Pro Asp Gln Pro Asn Gly
450 455 460 Val Ile Leu Asp Tyr Glu Leu Gln Tyr Tyr Glu Lys Glu Leu
Ser Glu 465 470 475 480 Tyr Asn Ala Thr Ala Ile Lys Ser Pro Thr Asn
Thr Val Thr Val Gln 485 490 495 Gly Leu Lys Ala Gly Ala Ile Tyr Val
Phe Gln Val Arg Ala Arg Thr 500 505 510 Val Ala Gly Tyr Gly Arg Tyr
Ser Gly Lys Met Tyr Phe Gln Thr Met 515 520 525 Thr Glu Ala Glu Tyr
Gln Thr Ser Ile Gln Glu Lys Leu Pro Leu Ile 530 535 540 Ile Gly Ser
Ser Ala Ala Gly Leu Val Phe Leu Ile Ala Val Val Val 545 550 555 560
Ile Ala Ile Val Cys Asn Arg Arg Arg Gly Phe Glu Arg Ala Asp Ser 565
570 575 Glu Tyr Thr Asp Lys Leu Gln His Tyr Thr Ser Gly His Met Thr
Pro 580 585 590 Gly Met Lys Ile Tyr Ile Asp Pro Phe Thr Tyr Glu Asp
Pro Asn Glu 595 600 605 Ala Val Arg Glu Phe Ala Lys Glu Ile Asp Ile
Ser Cys Val Lys Ile 610 615 620 Glu Gln Val Ile Gly Ala Gly Glu Phe
Gly Glu Val Cys Ser Gly His 625 630 635 640 Leu Lys Leu Pro Gly Lys
Arg Glu Ile Phe Val Ala Ile Lys Thr Leu 645 650 655 Lys Ser Gly Tyr
Thr Glu Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala 660 665 670 Ser Ile
Met Gly Gln Phe Asp His Pro Asn Val Ile His Leu Glu Gly 675 680 685
Val Val Thr Lys Ser Thr Pro Val Met Ile Ile Thr Glu Phe Met Glu 690
695 700 Asn Gly Ser Leu Asp Ser Phe Leu Arg Gln Asn Asp Gly Gln Phe
Thr 705 710 715 720 Val Ile Gln Leu Val Gly Met Leu Arg Gly Ile Ala
Ala Gly Met Lys 725 730 735 Tyr Leu Ala Asp Met Asn Tyr Val His Arg
Asp Leu Ala Ala Arg Asn 740 745 750 Ile Leu Val Asn Ser Asn Leu Val
Cys Lys Val Ser Asp Phe Gly Leu 755 760 765 Ser Arg Phe Leu Glu Asp
Asp Thr Ser Asp Pro Thr Tyr Thr Ser Ala 770 775 780 Leu Gly Gly Lys
Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Gln 785 790 795 800 Tyr
Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val 805 810
815 Met Trp Glu Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Thr
820 825 830 Asn Gln Asp Val Ile Asn Ala Ile Glu Gln Asp Tyr Arg Leu
Pro Pro 835 840 845 Pro Met Asp Cys Pro Ser Ala Leu His Gln Leu Met
Leu Asp Cys Trp 850 855 860 Gln Lys Asp Arg Asn His Arg Pro Lys Phe
Gly Gln Ile Val Asn Thr 865 870 875 880 Leu Asp Lys Met Ile Arg Asn
Pro Asn Ser Leu Lys Ala Met Ala Pro 885 890 895 Leu Ser Ser Gly Ile
Asn Leu Pro Leu Leu Asp Arg Thr Ile Pro Asp 900 905 910 Tyr Thr Ser
Phe Asn Thr Val Asp Glu Trp Leu Glu Ala Ile Lys Met 915 920 925 Gly
Gln Tyr Lys Glu Ser Phe Ala Asn Ala Gly Phe Thr Ser Phe Asp 930 935
940 Val Val Ser Gln Met Met Met Glu Asp Ile Leu Arg Val Gly Val Thr
945 950 955 960 Leu Ala Gly His Gln Lys Lys Ile Leu Asn Ser Ile Gln
Val Met Arg 965 970 975 Ala Gln Met Asn Gln Ile Gln Ser Val Glu Val
980 985 <210> SEQ ID NO 23 <211> LENGTH: 282
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: ASLG659
<400> SEQUENCE: 23 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp
Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala
Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr
Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly
Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp
Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80
His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85
90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly
Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala
Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Lys
Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro
Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu
Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val
Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val
Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205
Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210
215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys
Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln
Leu Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe
Ala Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met
Leu Lys 275 280 <210> SEQ ID NO 24 <211> LENGTH: 123
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: PSCA
<400> SEQUENCE: 24 Met Lys Ala Val Leu Leu Ala Leu Leu Met
Ala Gly Leu Ala Leu Gln 1 5 10 15 Pro Gly Thr Ala Leu Leu Cys Tyr
Ser Cys Lys Ala Gln Val Ser Asn 20 25 30 Glu Asp Cys Leu Gln Val
Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys 35 40 45 Trp Thr Ala Arg
Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys 50 55 60 Gly Cys
Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly 65 70 75 80
Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly 85
90 95 Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro
Ala 100 105 110 Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu 115 120
<210> SEQ ID NO 25
<211> LENGTH: 236 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
GEDA <400> SEQUENCE: 25 Met Pro Gly Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Met Leu 1 5 10 15 Pro Ala Gln Glu Ala Ala Lys
Leu Tyr His Thr Asn Tyr Val Arg Asn 20 25 30 Ser Arg Ala Ile Gly
Val Leu Trp Ala Ile Phe Thr Ile Cys Phe Ala 35 40 45 Ile Val Asn
Val Val Cys Phe Ile Gln Pro Tyr Trp Ile Gly Asp Gly 50 55 60 Val
Asp Thr Pro Gln Ala Gly Tyr Phe Gly Leu Phe His Tyr Cys Ile 65 70
75 80 Gly Asn Gly Phe Ser Arg Glu Leu Thr Cys Arg Gly Ser Phe Thr
Asp 85 90 95 Phe Ser Thr Leu Pro Ser Gly Ala Phe Lys Ala Ala Ser
Phe Phe Ile 100 105 110 Gly Leu Ser Met Met Leu Ile Ile Ala Cys Ile
Ile Cys Phe Thr Leu 115 120 125 Phe Phe Phe Cys Asn Thr Ala Thr Val
Tyr Lys Ile Cys Ala Trp Met 130 135 140 Gln Leu Thr Ser Ala Ala Cys
Leu Val Leu Gly Cys Met Ile Phe Pro 145 150 155 160 Asp Gly Trp Asp
Ser Asp Glu Val Lys Arg Met Cys Gly Glu Lys Thr 165 170 175 Asp Lys
Tyr Thr Leu Gly Ala Cys Ser Val Arg Trp Ala Tyr Ile Leu 180 185 190
Ala Ile Ile Gly Ile Leu Asp Ala Leu Ile Leu Ser Phe Leu Ala Phe 195
200 205 Val Leu Gly Asn Arg Gln Asp Ser Leu Met Ala Glu Glu Leu Lys
Ala 210 215 220 Glu Asn Lys Val Leu Leu Ser Gln Tyr Ser Leu Glu 225
230 235 <210> SEQ ID NO 26 <211> LENGTH: 184
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: BAFF-R
<400> SEQUENCE: 26 Met Arg Arg Gly Pro Arg Ser Leu Arg Gly
Arg Asp Ala Pro Ala Pro 1 5 10 15 Thr Pro Cys Val Pro Ala Glu Cys
Phe Asp Leu Leu Val Arg His Cys 20 25 30 Val Ala Cys Gly Leu Leu
Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala 35 40 45 Ser Ser Pro Ala
Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val Gly 50 55 60 Ala Gly
Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu Phe Gly 65 70 75 80
Ala Pro Ala Leu Leu Gly Leu Ala Leu Val Leu Ala Leu Val Leu Val 85
90 95 Gly Leu Val Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala
Ser 100 105 110 Ser Ala Glu Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu
Pro Leu Asp 115 120 125 Lys Val Ile Ile Leu Ser Pro Gly Ile Ser Asp
Ala Thr Ala Pro Ala 130 135 140 Trp Pro Pro Pro Gly Glu Asp Pro Gly
Thr Thr Pro Pro Gly His Ser 145 150 155 160 Val Pro Val Pro Ala Thr
Glu Leu Gly Ser Thr Glu Leu Val Thr Thr 165 170 175 Lys Thr Ala Gly
Pro Glu Gln Gln 180 <210> SEQ ID NO 27 <211> LENGTH:
847 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: CD22
<400> SEQUENCE: 27 Met His Leu Leu Gly Pro Trp Leu Leu Leu
Leu Val Leu Glu Tyr Leu 1 5 10 15 Ala Phe Ser Asp Ser Ser Lys Trp
Val Phe Glu His Pro Glu Thr Leu 20 25 30 Tyr Ala Trp Glu Gly Ala
Cys Val Trp Ile Pro Cys Thr Tyr Arg Ala 35 40 45 Leu Asp Gly Asp
Leu Glu Ser Phe Ile Leu Phe His Asn Pro Glu Tyr 50 55 60 Asn Lys
Asn Thr Ser Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr 65 70 75 80
Lys Asp Gly Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly 85
90 95 Asp Lys Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu
Asn 100 105 110 Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr
Glu Lys Trp 115 120 125 Met Glu Arg Ile His Leu Asn Val Ser Glu Arg
Pro Phe Pro Pro His 130 135 140 Ile Gln Leu Pro Pro Glu Ile Gln Glu
Ser Gln Glu Val Thr Leu Thr 145 150 155 160 Cys Leu Leu Asn Phe Ser
Cys Tyr Gly Tyr Pro Ile Gln Leu Gln Trp 165 170 175 Leu Leu Glu Gly
Val Pro Met Arg Gln Ala Ala Val Thr Ser Thr Ser 180 185 190 Leu Thr
Ile Lys Ser Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro 195 200 205
Gln Trp Ser His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala 210
215 220 Asp Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys
His 225 230 235 240 Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp
Ala Ile Val Arg 245 250 255 Glu Gly Asp Ser Val Thr Met Thr Cys Glu
Val Ser Ser Ser Asn Pro 260 265 270 Glu Tyr Thr Thr Val Ser Trp Leu
Lys Asp Gly Thr Ser Leu Lys Lys 275 280 285 Gln Asn Thr Phe Thr Leu
Asn Leu Arg Glu Val Thr Lys Asp Gln Ser 290 295 300 Gly Lys Tyr Cys
Cys Gln Val Ser Asn Asp Val Gly Pro Gly Arg Ser 305 310 315 320 Glu
Glu Val Phe Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val 325 330
335 Gln Ile Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu
340 345 350 Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp
Tyr His 355 360 365 Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys
Val His Ile Pro 370 375 380 Lys Ile Leu Pro Trp His Ala Gly Thr Tyr
Ser Cys Val Ala Glu Asn 385 390 395 400 Ile Leu Gly Thr Gly Gln Arg
Gly Pro Gly Ala Glu Leu Asp Val Gln 405 410 415 Tyr Pro Pro Lys Lys
Val Thr Thr Val Ile Gln Asn Pro Met Pro Ile 420 425 430 Arg Glu Gly
Asp Thr Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn 435 440 445 Pro
Ser Val Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu 450 455
460 Pro Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr
465 470 475 480 Thr Ile Ala Cys Ala Arg Cys Asn Ser Trp Cys Ser Trp
Ala Ser Pro 485 490 495 Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp
Val Arg Val Arg Lys 500 505 510 Ile Lys Pro Leu Ser Glu Ile His Ser
Gly Asn Ser Val Ser Leu Gln 515 520 525 Cys Asp Phe Ser Ser Ser His
Pro Lys Glu Val Gln Phe Phe Trp Glu 530 535 540 Lys Asn Gly Arg Leu
Leu Gly Lys Glu Ser Gln Leu Asn Phe Asp Ser 545 550 555 560 Ile Ser
Pro Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser 565 570 575
Ile Gly Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala 580
585 590 Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met
Glu 595 600 605 Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn
Pro Pro Val 610 615 620 Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln
Ser Leu Pro His His 625 630 635 640 Ser Gln Lys Leu Arg Leu Glu Pro
Val Lys Val Gln His Ser Gly Ala 645 650 655 Tyr Trp Cys Gln Gly Thr
Asn Ser Val Gly Lys Gly Arg Ser Pro Leu 660 665 670 Ser Thr Leu Thr
Val Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Val 675 680 685 Ala Val
Gly Leu Gly Ser Cys Leu Ala Ile Leu Ile Leu Ala Ile Cys 690 695 700
Gly Leu Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly 705
710 715 720 Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn
Lys Lys 725 730 735 Val Arg Arg Ala Pro Leu Ser Glu Gly Pro His Ser
Leu Gly Cys Tyr 740 745 750 Asn Pro Met Met Glu Asp Gly Ile Ser Tyr
Thr Thr Leu Arg Phe Pro
755 760 765 Glu Met Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu
Met Gln 770 775 780 Arg Pro Pro Arg Thr Cys Asp Asp Thr Val Thr Tyr
Ser Ala Leu His 785 790 795 800 Lys Arg Gln Val Gly Asp Tyr Glu Asn
Val Ile Pro Asp Phe Pro Glu 805 810 815 Asp Glu Gly Ile His Tyr Ser
Glu Leu Ile Gln Phe Gly Val Gly Glu 820 825 830 Arg Pro Gln Ala Gln
Glu Asn Val Asp Tyr Val Ile Leu Lys His 835 840 845 <210> SEQ
ID NO 28 <211> LENGTH: 226 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: CD79A <400> SEQUENCE: 28 Met Pro Gly Gly Pro Gly
Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu
Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp
Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45
Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50
55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu
Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile
Gln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg
Val Gln Glu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr
Tyr Leu Arg Val Arg Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp
Met Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly
Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly 145 150 155 160 Thr Leu
Leu Leu Phe Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175
Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180
185 190 Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln
Gly 195 200 205 Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val
Gln Leu Glu 210 215 220 Lys Pro 225 <210> SEQ ID NO 29
<211> LENGTH: 372 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
CXCR5 <400> SEQUENCE: 29 Met Asn Tyr Pro Leu Thr Leu Glu Met
Asp Leu Glu Asn Leu Glu Asp 1 5 10 15 Leu Phe Trp Glu Leu Asp Arg
Leu Asp Asn Tyr Asn Asp Thr Ser Leu 20 25 30 Val Glu Asn His Leu
Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser 35 40 45 Phe Lys Ala
Val Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu 50 55 60 Gly
Val Ile Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg 65 70
75 80 Gln Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val
Ala 85 90 95 Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala
Glu Gly Ser 100 105 110 Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys
Thr Val Ile Ala Leu 115 120 125 His Lys Val Asn Phe Tyr Cys Ser Ser
Leu Leu Leu Ala Cys Ile Ala 130 135 140 Val Asp Arg Tyr Leu Ala Ile
Val His Ala Val His Ala Tyr Arg His 145 150 155 160 Arg Arg Leu Leu
Ser Ile His Ile Thr Cys Gly Thr Ile Trp Leu Val 165 170 175 Gly Phe
Leu Leu Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln 180 185 190
Gly His His Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn 195
200 205 Gln Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His
Val 210 215 220 Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys
Tyr Val Gly 225 230 235 240 Val Val His Arg Leu Arg Gln Ala Gln Arg
Arg Pro Gln Arg Gln Lys 245 250 255 Ala Val Arg Val Ala Ile Leu Val
Thr Ser Ile Phe Phe Leu Cys Trp 260 265 270 Ser Pro Tyr His Ile Val
Ile Phe Leu Asp Thr Leu Ala Arg Leu Lys 275 280 285 Ala Val Asp Asn
Thr Cys Lys Leu Asn Gly Ser Leu Pro Val Ala Ile 290 295 300 Thr Met
Cys Glu Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met 305 310 315
320 Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu
325 330 335 Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln
Leu Phe 340 345 350 Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu
Asn Ala Thr Ser 355 360 365 Leu Thr Thr Phe 370 <210> SEQ ID
NO 30 <211> LENGTH: 273 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: HLA-DOB <400> SEQUENCE: 30 Met Gly Ser Gly Trp
Val Pro Trp Val Val Ala Leu Leu Val Asn Leu 1 5 10 15 Thr Arg Leu
Asp Ser Ser Met Thr Gln Gly Thr Asp Ser Pro Glu Asp 20 25 30 Phe
Val Ile Gln Ala Lys Ala Asp Cys Tyr Phe Thr Asn Gly Thr Glu 35 40
45 Lys Val Gln Phe Val Val Arg Phe Ile Phe Asn Leu Glu Glu Tyr Val
50 55 60 Arg Phe Asp Ser Asp Val Gly Met Phe Val Ala Leu Thr Lys
Leu Gly 65 70 75 80 Gln Pro Asp Ala Glu Gln Trp Asn Ser Arg Leu Asp
Leu Leu Glu Arg 85 90 95 Ser Arg Gln Ala Val Asp Gly Val Cys Arg
His Asn Tyr Arg Leu Gly 100 105 110 Ala Pro Phe Thr Val Gly Arg Lys
Val Gln Pro Glu Val Thr Val Tyr 115 120 125 Pro Glu Arg Thr Pro Leu
Leu His Gln His Asn Leu Leu His Cys Ser 130 135 140 Val Thr Gly Phe
Tyr Pro Gly Asp Ile Lys Ile Lys Trp Phe Leu Asn 145 150 155 160 Gly
Gln Glu Glu Arg Ala Gly Val Met Ser Thr Gly Pro Ile Arg Asn 165 170
175 Gly Asp Trp Thr Phe Gln Thr Val Val Met Leu Glu Met Thr Pro Glu
180 185 190 Leu Gly His Val Tyr Thr Cys Leu Val Asp His Ser Ser Leu
Leu Ser 195 200 205 Pro Val Ser Val Glu Trp Arg Ala Gln Ser Glu Tyr
Ser Trp Arg Lys 210 215 220 Met Leu Ser Gly Ile Ala Ala Phe Leu Leu
Gly Leu Ile Phe Leu Leu 225 230 235 240 Val Gly Ile Val Ile Gln Leu
Arg Ala Gln Lys Gly Tyr Val Arg Thr 245 250 255 Gln Met Ser Gly Asn
Glu Val Ser Arg Ala Val Leu Leu Pro Gln Ser 260 265 270 Cys
<210> SEQ ID NO 31 <211> LENGTH: 422 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: Purinergic receptor P2X ligand-gated
ion channel 5 <400> SEQUENCE: 31 Met Gly Gln Ala Gly Cys Lys
Gly Leu Cys Leu Ser Leu Phe Asp Tyr 1 5 10 15 Lys Thr Glu Lys Tyr
Val Ile Ala Lys Asn Lys Lys Val Gly Leu Leu 20 25 30 Tyr Arg Leu
Leu Gln Ala Ser Ile Leu Ala Tyr Leu Val Val Trp Val 35 40 45 Phe
Leu Ile Lys Lys Gly Tyr Gln Asp Val Asp Thr Ser Leu Gln Ser 50 55
60 Ala Val Ile Thr Lys Val Lys Gly Val Ala Phe Thr Asn Thr Ser Asp
65 70 75 80 Leu Gly Gln Arg Ile Trp Asp Val Ala Asp Tyr Val Ile Pro
Ala Gln 85 90 95 Gly Glu Asn Val Phe Phe Val Val Thr Asn Leu Ile
Val Thr Pro Asn 100 105 110 Gln Arg Gln Asn Val Cys Ala Glu Asn Glu
Gly Ile Pro Asp Gly Ala 115 120 125
Cys Ser Lys Asp Ser Asp Cys His Ala Gly Glu Ala Val Thr Ala Gly 130
135 140 Asn Gly Val Lys Thr Gly Arg Cys Leu Arg Arg Glu Asn Leu Ala
Arg 145 150 155 160 Gly Thr Cys Glu Ile Phe Ala Trp Cys Pro Leu Glu
Thr Ser Ser Arg 165 170 175 Pro Glu Glu Pro Phe Leu Lys Glu Ala Glu
Asp Phe Thr Ile Phe Ile 180 185 190 Lys Asn His Ile Arg Phe Pro Lys
Phe Asn Phe Ser Lys Ser Asn Val 195 200 205 Met Asp Val Lys Asp Arg
Ser Phe Leu Lys Ser Cys His Phe Gly Pro 210 215 220 Lys Asn His Tyr
Cys Pro Ile Phe Arg Leu Gly Ser Val Ile Arg Trp 225 230 235 240 Ala
Gly Ser Asp Phe Gln Asp Ile Ala Leu Glu Gly Gly Val Ile Gly 245 250
255 Ile Asn Ile Glu Trp Asn Cys Asp Leu Asp Lys Ala Ala Ser Glu Cys
260 265 270 His Pro His Tyr Ser Phe Ser Arg Leu Asp Asn Lys Leu Ser
Lys Ser 275 280 285 Val Ser Ser Gly Tyr Asn Phe Arg Phe Ala Arg Tyr
Tyr Arg Asp Ala 290 295 300 Ala Gly Val Glu Phe Arg Thr Leu Met Lys
Ala Tyr Gly Ile Arg Phe 305 310 315 320 Asp Val Met Val Asn Gly Lys
Gly Ala Phe Phe Cys Asp Leu Val Leu 325 330 335 Ile Tyr Leu Ile Lys
Lys Arg Glu Phe Tyr Arg Asp Lys Lys Tyr Glu 340 345 350 Glu Val Arg
Gly Leu Glu Asp Ser Ser Gln Glu Ala Glu Asp Glu Ala 355 360 365 Ser
Gly Leu Gly Leu Ser Glu Gln Leu Thr Ser Gly Pro Gly Leu Leu 370 375
380 Gly Met Pro Glu Gln Gln Glu Leu Gln Glu Pro Pro Glu Ala Lys Arg
385 390 395 400 Gly Ser Ser Ser Gln Lys Gly Asn Gly Ser Val Cys Pro
Gln Leu Leu 405 410 415 Glu Pro His Arg Ser Thr 420 <210> SEQ
ID NO 32 <211> LENGTH: 359 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: CD72 <400> SEQUENCE: 32 Met Ala Glu Ala Ile Thr
Tyr Ala Asp Leu Arg Phe Val Lys Ala Pro 1 5 10 15 Leu Lys Lys Ser
Ile Ser Ser Arg Leu Gly Gln Asp Pro Gly Ala Asp 20 25 30 Asp Asp
Gly Glu Ile Thr Tyr Glu Asn Val Gln Val Pro Ala Val Leu 35 40 45
Gly Val Pro Ser Ser Leu Ala Ser Ser Val Leu Gly Asp Lys Ala Ala 50
55 60 Val Lys Ser Glu Gln Pro Thr Ala Ser Trp Arg Ala Val Thr Ser
Pro 65 70 75 80 Ala Val Gly Arg Ile Leu Pro Cys Arg Thr Thr Cys Leu
Arg Tyr Leu 85 90 95 Leu Leu Gly Leu Leu Leu Thr Cys Leu Leu Leu
Gly Val Thr Ala Ile 100 105 110 Cys Leu Gly Val Arg Tyr Leu Gln Val
Ser Gln Gln Leu Gln Gln Thr 115 120 125 Asn Arg Val Leu Glu Val Thr
Asn Ser Ser Leu Arg Gln Gln Leu Arg 130 135 140 Leu Lys Ile Thr Gln
Leu Gly Gln Ser Ala Glu Asp Leu Gln Gly Ser 145 150 155 160 Arg Arg
Glu Leu Ala Gln Ser Gln Glu Ala Leu Gln Val Glu Gln Arg 165 170 175
Ala His Gln Ala Ala Glu Gly Gln Leu Gln Ala Cys Gln Ala Asp Arg 180
185 190 Gln Lys Thr Lys Glu Thr Leu Gln Ser Glu Glu Gln Gln Arg Arg
Ala 195 200 205 Leu Glu Gln Lys Leu Ser Asn Met Glu Asn Arg Leu Lys
Pro Phe Phe 210 215 220 Thr Cys Gly Ser Ala Asp Thr Cys Cys Pro Ser
Gly Trp Ile Met His 225 230 235 240 Gln Lys Ser Cys Phe Tyr Ile Ser
Leu Thr Ser Lys Asn Trp Gln Glu 245 250 255 Ser Gln Lys Gln Cys Glu
Thr Leu Ser Ser Lys Leu Ala Thr Phe Ser 260 265 270 Glu Ile Tyr Pro
Gln Ser His Ser Tyr Tyr Phe Leu Asn Ser Leu Leu 275 280 285 Pro Asn
Gly Gly Ser Gly Asn Ser Tyr Trp Thr Gly Leu Ser Ser Asn 290 295 300
Lys Asp Trp Lys Leu Thr Asp Asp Thr Gln Arg Thr Arg Thr Tyr Ala 305
310 315 320 Gln Ser Ser Lys Cys Asn Lys Val His Lys Thr Trp Ser Trp
Trp Thr 325 330 335 Leu Glu Ser Glu Ser Cys Arg Ser Ser Leu Pro Tyr
Ile Cys Glu Met 340 345 350 Thr Ala Phe Arg Phe Pro Asp 355
<210> SEQ ID NO 33 <211> LENGTH: 661 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: LY64 <400> SEQUENCE: 33 Met
Ala Phe Asp Val Ser Cys Phe Phe Trp Val Val Leu Phe Ser Ala 1 5 10
15 Gly Cys Lys Val Ile Thr Ser Trp Asp Gln Met Cys Ile Glu Lys Glu
20 25 30 Ala Asn Lys Thr Tyr Asn Cys Glu Asn Leu Gly Leu Ser Glu
Ile Pro 35 40 45 Asp Thr Leu Pro Asn Thr Thr Glu Phe Leu Glu Phe
Ser Phe Asn Phe 50 55 60 Leu Pro Thr Ile His Asn Arg Thr Phe Ser
Arg Leu Met Asn Leu Thr 65 70 75 80 Phe Leu Asp Leu Thr Arg Cys Gln
Ile Asn Trp Ile His Glu Asp Thr 85 90 95 Phe Gln Ser His His Gln
Leu Ser Thr Leu Val Leu Thr Gly Asn Pro 100 105 110 Leu Ile Phe Met
Ala Glu Thr Ser Leu Asn Gly Pro Lys Ser Leu Lys 115 120 125 His Leu
Phe Leu Ile Gln Thr Gly Ile Ser Asn Leu Glu Phe Ile Pro 130 135 140
Val His Asn Leu Glu Asn Leu Glu Ser Leu Tyr Leu Gly Ser Asn His 145
150 155 160 Ile Ser Ser Ile Lys Phe Pro Lys Asp Phe Pro Ala Arg Asn
Leu Lys 165 170 175 Val Leu Asp Phe Gln Asn Asn Ala Ile His Tyr Ile
Ser Arg Glu Asp 180 185 190 Met Arg Ser Leu Glu Gln Ala Ile Asn Leu
Ser Leu Asn Phe Asn Gly 195 200 205 Asn Asn Val Lys Gly Ile Glu Leu
Gly Ala Phe Asp Ser Thr Val Phe 210 215 220 Gln Ser Leu Asn Phe Gly
Gly Thr Pro Asn Leu Ser Val Ile Phe Asn 225 230 235 240 Gly Leu Gln
Asn Ser Thr Thr Gln Ser Leu Trp Leu Gly Thr Phe Glu 245 250 255 Asp
Ile Asp Asp Glu Asp Ile Ser Ser Ala Met Leu Lys Gly Leu Cys 260 265
270 Glu Met Ser Val Glu Ser Leu Asn Leu Gln Glu His Arg Phe Ser Asp
275 280 285 Ile Ser Ser Thr Thr Phe Gln Cys Phe Thr Gln Leu Gln Glu
Leu Asp 290 295 300 Leu Thr Ala Thr His Leu Lys Gly Leu Pro Ser Gly
Met Lys Gly Leu 305 310 315 320 Asn Leu Leu Lys Lys Leu Val Leu Ser
Val Asn His Phe Asp Gln Leu 325 330 335 Cys Gln Ile Ser Ala Ala Asn
Phe Pro Ser Leu Thr His Leu Tyr Ile 340 345 350 Arg Gly Asn Val Lys
Lys Leu His Leu Gly Val Gly Cys Leu Glu Lys 355 360 365 Leu Gly Asn
Leu Gln Thr Leu Asp Leu Ser His Asn Asp Ile Glu Ala 370 375 380 Ser
Asp Cys Cys Ser Leu Gln Leu Lys Asn Leu Ser His Leu Gln Thr 385 390
395 400 Leu Asn Leu Ser His Asn Glu Pro Leu Gly Leu Gln Ser Gln Ala
Phe 405 410 415 Lys Glu Cys Pro Gln Leu Glu Leu Leu Asp Leu Ala Phe
Thr Arg Leu 420 425 430 His Ile Asn Ala Pro Gln Ser Pro Phe Gln Asn
Leu His Phe Leu Gln 435 440 445 Val Leu Asn Leu Thr Tyr Cys Phe Leu
Asp Thr Ser Asn Gln His Leu 450 455 460 Leu Ala Gly Leu Pro Val Leu
Arg His Leu Asn Leu Lys Gly Asn His 465 470 475 480 Phe Gln Asp Gly
Thr Ile Thr Lys Thr Asn Leu Leu Gln Thr Val Gly 485 490 495 Ser Leu
Glu Val Leu Ile Leu Ser Ser Cys Gly Leu Leu Ser Ile Asp 500 505 510
Gln Gln Ala Phe His Ser Leu Gly Lys Met Ser His Val Asp Leu Ser 515
520 525 His Asn Ser Leu Thr Cys Asp Ser Ile Asp Ser Leu Ser His Leu
Lys 530 535 540 Gly Ile Tyr Leu Asn Leu Ala Ala Asn Ser Ile Asn Ile
Ile Ser Pro 545 550 555 560 Arg Leu Leu Pro Ile Leu Ser Gln Gln Ser
Thr Ile Asn Leu Ser His
565 570 575 Asn Pro Leu Asp Cys Thr Cys Ser Asn Ile His Phe Leu Thr
Trp Tyr 580 585 590 Lys Glu Asn Leu His Lys Leu Glu Gly Ser Glu Glu
Thr Thr Cys Ala 595 600 605 Asn Pro Pro Ser Leu Arg Gly Val Lys Leu
Ser Asp Val Lys Leu Ser 610 615 620 Cys Gly Ile Thr Ala Ile Gly Ile
Phe Phe Leu Ile Val Phe Leu Leu 625 630 635 640 Leu Leu Ala Ile Leu
Leu Phe Phe Ala Val Lys Tyr Leu Leu Arg Trp 645 650 655 Lys Tyr Gln
His Ile 660 <210> SEQ ID NO 34 <211> LENGTH: 429
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: FCRH1
<400> SEQUENCE: 34 Met Leu Pro Arg Leu Leu Leu Leu Ile Cys
Ala Pro Leu Cys Glu Pro 1 5 10 15 Ala Glu Leu Phe Leu Ile Ala Ser
Pro Ser His Pro Thr Glu Gly Ser 20 25 30 Pro Val Thr Leu Thr Cys
Lys Met Pro Phe Leu Gln Ser Ser Asp Ala 35 40 45 Gln Phe Gln Phe
Cys Phe Phe Arg Asp Thr Arg Ala Leu Gly Pro Gly 50 55 60 Trp Ser
Ser Ser Pro Lys Leu Gln Ile Ala Ala Met Trp Lys Glu Asp 65 70 75 80
Thr Gly Ser Tyr Trp Cys Glu Ala Gln Thr Met Ala Ser Lys Val Leu 85
90 95 Arg Ser Arg Arg Ser Gln Ile Asn Val His Arg Val Pro Val Ala
Asp 100 105 110 Val Ser Leu Glu Thr Gln Pro Pro Gly Gly Gln Val Met
Glu Gly Asp 115 120 125 Arg Leu Val Leu Ile Cys Ser Val Ala Met Gly
Thr Gly Asp Ile Thr 130 135 140 Phe Leu Trp Tyr Lys Gly Ala Val Gly
Leu Asn Leu Gln Ser Lys Thr 145 150 155 160 Gln Arg Ser Leu Thr Ala
Glu Tyr Glu Ile Pro Ser Val Arg Glu Ser 165 170 175 Asp Ala Glu Gln
Tyr Tyr Cys Val Ala Glu Asn Gly Tyr Gly Pro Ser 180 185 190 Pro Ser
Gly Leu Val Ser Ile Thr Val Arg Ile Pro Val Ser Arg Pro 195 200 205
Ile Leu Met Leu Arg Ala Pro Arg Ala Gln Ala Ala Val Glu Asp Val 210
215 220 Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Pro Pro Ile Leu
Tyr 225 230 235 240 Trp Phe Tyr His Glu Asp Ile Thr Leu Gly Ser Arg
Ser Ala Pro Ser 245 250 255 Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu
Thr Glu Glu His Ser Gly 260 265 270 Asn Tyr Ser Cys Glu Ala Asn Asn
Gly Leu Gly Ala Gln Arg Ser Glu 275 280 285 Ala Val Thr Leu Asn Phe
Thr Val Pro Thr Gly Ala Arg Ser Asn His 290 295 300 Leu Thr Ser Gly
Val Ile Glu Gly Leu Leu Ser Thr Leu Gly Pro Ala 305 310 315 320 Thr
Val Ala Leu Leu Phe Cys Tyr Gly Leu Lys Arg Lys Ile Gly Arg 325 330
335 Arg Ser Ala Arg Asp Pro Leu Arg Ser Leu Pro Ser Pro Leu Pro Gln
340 345 350 Glu Phe Thr Tyr Leu Asn Ser Pro Thr Pro Gly Gln Leu Gln
Pro Ile 355 360 365 Tyr Glu Asn Val Asn Val Val Ser Gly Asp Glu Val
Tyr Ser Leu Ala 370 375 380 Tyr Tyr Asn Gln Pro Glu Gln Glu Ser Val
Ala Ala Glu Thr Leu Gly 385 390 395 400 Thr His Met Glu Asp Lys Val
Ser Leu Asp Ile Tyr Ser Arg Leu Arg 405 410 415 Lys Ala Asn Ile Thr
Asp Val Asp Tyr Glu Asp Ala Met 420 425 <210> SEQ ID NO 35
<211> LENGTH: 977 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
IRTA2 <400> SEQUENCE: 35 Met Leu Leu Trp Val Ile Leu Leu Val
Leu Ala Pro Val Ser Gly Gln 1 5 10 15 Phe Ala Arg Thr Pro Arg Pro
Ile Ile Phe Leu Gln Pro Pro Trp Thr 20 25 30 Thr Val Phe Gln Gly
Glu Arg Val Thr Leu Thr Cys Lys Gly Phe Arg 35 40 45 Phe Tyr Ser
Pro Gln Lys Thr Lys Trp Tyr His Arg Tyr Leu Gly Lys 50 55 60 Glu
Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu Glu Val Gln Glu Ser 65 70
75 80 Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser Pro Leu Ser Ser Pro
Val 85 90 95 His Leu Asp Phe Ser Ser Ala Ser Leu Ile Leu Gln Ala
Pro Leu Ser 100 105 110 Val Phe Glu Gly Asp Ser Val Val Leu Arg Cys
Arg Ala Lys Ala Glu 115 120 125 Val Thr Leu Asn Asn Thr Ile Tyr Lys
Asn Asp Asn Val Leu Ala Phe 130 135 140 Leu Asn Lys Arg Thr Asp Phe
His Ile Pro His Ala Cys Leu Lys Asp 145 150 155 160 Asn Gly Ala Tyr
Arg Cys Thr Gly Tyr Lys Glu Ser Cys Cys Pro Val 165 170 175 Ser Ser
Asn Thr Val Lys Ile Gln Val Gln Glu Pro Phe Thr Arg Pro 180 185 190
Val Leu Arg Ala Ser Ser Phe Gln Pro Ile Ser Gly Asn Pro Val Thr 195
200 205 Leu Thr Cys Glu Thr Gln Leu Ser Leu Glu Arg Ser Asp Val Pro
Leu 210 215 220 Arg Phe Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu
Gly Trp Ser 225 230 235 240 Leu Ser Pro Asn Phe Gln Ile Thr Ala Met
Trp Ser Lys Asp Ser Gly 245 250 255 Phe Tyr Trp Cys Lys Ala Ala Thr
Met Pro His Ser Val Ile Ser Asp 260 265 270 Ser Pro Arg Ser Trp Ile
Gln Val Gln Ile Pro Ala Ser His Pro Val 275 280 285 Leu Thr Leu Ser
Pro Glu Lys Ala Leu Asn Phe Glu Gly Thr Lys Val 290 295 300 Thr Leu
His Cys Glu Thr Gln Glu Asp Ser Leu Arg Thr Leu Tyr Arg 305 310 315
320 Phe Tyr His Glu Gly Val Pro Leu Arg His Lys Ser Val Arg Cys Glu
325 330 335 Arg Gly Ala Ser Ile Ser Phe Ser Leu Thr Thr Glu Asn Ser
Gly Asn 340 345 350 Tyr Tyr Cys Thr Ala Asp Asn Gly Leu Gly Ala Lys
Pro Ser Lys Ala 355 360 365 Val Ser Leu Ser Val Thr Val Pro Val Ser
His Pro Val Leu Asn Leu 370 375 380 Ser Ser Pro Glu Asp Leu Ile Phe
Glu Gly Ala Lys Val Thr Leu His 385 390 395 400 Cys Glu Ala Gln Arg
Gly Ser Leu Pro Ile Leu Tyr Gln Phe His His 405 410 415 Glu Asp Ala
Ala Leu Glu Arg Arg Ser Ala Asn Ser Ala Gly Gly Val 420 425 430 Ala
Ile Ser Phe Ser Leu Thr Ala Glu His Ser Gly Asn Tyr Tyr Cys 435 440
445 Thr Ala Asp Asn Gly Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu
450 455 460 Ser Ile Thr Val Pro Val Ser His Pro Val Leu Thr Leu Ser
Ser Ala 465 470 475 480 Glu Ala Leu Thr Phe Glu Gly Ala Thr Val Thr
Leu His Cys Glu Val 485 490 495 Gln Arg Gly Ser Pro Gln Ile Leu Tyr
Gln Phe Tyr His Glu Asp Met 500 505 510 Pro Leu Trp Ser Ser Ser Thr
Pro Ser Val Gly Arg Val Ser Phe Ser 515 520 525 Phe Ser Leu Thr Glu
Gly His Ser Gly Asn Tyr Tyr Cys Thr Ala Asp 530 535 540 Asn Gly Phe
Gly Pro Gln Arg Ser Glu Val Val Ser Leu Phe Val Thr 545 550 555 560
Val Pro Val Ser Arg Pro Ile Leu Thr Leu Arg Val Pro Arg Ala Gln 565
570 575 Ala Val Val Gly Asp Leu Leu Glu Leu His Cys Glu Ala Pro Arg
Gly 580 585 590 Ser Pro Pro Ile Leu Tyr Trp Phe Tyr His Glu Asp Val
Thr Leu Gly 595 600 605 Ser Ser Ser Ala Pro Ser Gly Gly Glu Ala Ser
Phe Asn Leu Ser Leu 610 615 620 Thr Ala Glu His Ser Gly Asn Tyr Ser
Cys Glu Ala Asn Asn Gly Leu 625 630 635 640 Val Ala Gln His Ser Asp
Thr Ile Ser Leu Ser Val Ile Val Pro Val 645 650 655 Ser Arg Pro Ile
Leu Thr Phe Arg Ala Pro Arg Ala Gln Ala Val Val 660 665 670 Gly Asp
Leu Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Ser Pro 675 680 685
Ile Leu Tyr Trp Phe Tyr His Glu Asp Val Thr Leu Gly Lys Ile Ser 690
695 700 Ala Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr
Glu 705 710 715 720 His Ser Gly Ile Tyr Ser Cys Glu Ala Asp Asn Gly
Pro Glu Ala Gln 725 730 735 Arg Ser Glu Met Val Thr Leu Lys Val Ala
Val Pro Val Ser Arg Pro 740 745 750 Val Leu Thr Leu Arg Ala Pro Gly
Thr His Ala Ala Val Gly Asp Leu 755 760 765 Leu Glu Leu His Cys Glu
Ala Leu Arg Gly Ser Pro Leu Ile Leu Tyr 770 775 780 Arg Phe Phe His
Glu Asp Val Thr Leu Gly Asn Arg Ser Ser Pro Ser 785 790 795 800 Gly
Gly Ala Ser Leu Asn Leu Ser Leu Thr Ala Glu His Ser Gly Asn 805 810
815 Tyr Ser Cys Glu Ala Asp Asn Gly Leu Gly Ala Gln Arg Ser Glu Thr
820 825 830 Val Thr Leu Tyr Ile Thr Gly Leu Thr Ala Asn Arg Ser Gly
Pro Phe 835 840 845
Ala Thr Gly Val Ala Gly Gly Leu Leu Ser Ile Ala Gly Leu Ala Ala 850
855 860 Gly Ala Leu Leu Leu Tyr Cys Trp Leu Ser Arg Lys Ala Gly Arg
Lys 865 870 875 880 Pro Ala Ser Asp Pro Ala Arg Ser Pro Pro Asp Ser
Asp Ser Gln Glu 885 890 895 Pro Thr Tyr His Asn Val Pro Ala Trp Glu
Glu Leu Gln Pro Val Tyr 900 905 910 Thr Asn Ala Asn Pro Arg Gly Glu
Asn Val Val Tyr Ser Glu Val Arg 915 920 925 Ile Ile Gln Glu Lys Lys
Lys His Ala Val Ala Ser Asp Pro Arg His 930 935 940 Leu Arg Asn Lys
Gly Ser Pro Ile Ile Tyr Ser Glu Val Lys Val Ala 945 950 955 960 Ser
Thr Pro Val Ser Gly Ser Leu Phe Leu Ala Ser Ser Ala Pro His 965 970
975 Arg
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